Bont/a peptides and methodsof predicting and reducing immunoresistance to botulinum toxin therapy

ABSTRACT

The present invention provides BoNT/A peptides as well as methods of predicting or determining immunoresistance to botulinum toxin therapy in an individual using BoNT/A peptides.

This application is a divisional application and claims prioritypursuant to 35 U.S.C. §120 to U.S. patent application Ser. No.11/693,111, filed Mar. 29, 2007, now U.S. Pat. No. 7,635,484 acontinuation that claims priority pursuant to 35 U.S.C. §120 to U.S.patent application Ser. No. 10/821,669, filed Apr. 9, 2004, now U.S.Pat. No. 7,341,843, each of which is hereby incorporated by reference inits entirety.

FIELD OF INVENTION

This invention relates generally to the field of immunology, and, morespecifically, to the use of botulinum toxin peptides and anti-botulinumtoxin antibodies as diagnostic and therapeutic agents.

BACKGROUND OF THE INVENTION

Botulinum neurotoxins are proteins produced by several strains of thebacterium Clostridium botulinum, the spores of which are abundant insoil and marine sediments. These proteins are the most toxic substancesknown to man, being more lethal per molecule than diphtheria toxin,curare and sodium cyanide. There are seven distinct but relatedbotulinum toxin serotypes, designated A through G. Botulinum toxin typesA, B, E, and F are the most common causes of botulism in humans, whiletypes C and D cause botulism in other mammals and birds. All sevenbotulinum toxin serotypes act by similar mechanisms and produce similarlethal effects when inhaled or ingested.

Botulinum toxins interrupt signals normally transmitted from nerve tomuscle, thereby resulting in paralysis. Normally, electrical impulsesthat control muscle function are generated by the brain, brain stem andspinal cord, and these impulses travel from the originating area intoperipheral nerves, which control motor function. At the end of theseperipheral nerves are compartments for the neurotransmitteracetylcholine, a chemical messenger that transmits the electrical signalof the peripheral nerve to the muscle, instructing the muscle tocontract. In the absence of botulinum toxin, acetylcholine is releasedinto the junction between peripheral nerve and muscle when an electricalimpulse reaches the storage compartment. The released acetylcholinebinds to receptors located on the muscle, signaling the ensuing musclecontraction. However, botulinum toxin interferes with the release ofacetylcholine into the junction, thereby blocking transmission of theelectrical signal. Normal muscular contraction terminates due to theabsence of the electrical signal.

In spite of their potentially deleterious effects, the ability of low,controlled doses of botulinum toxins to block acetylcholine release isuseful in treating conditions characterized by unwanted muscularcontraction or spasm resulting from excessive neural activity. Over thepast 10 years, botulinum toxins have emerged as an important therapeutictool for a number of neurological and ophthalmic conditions that havefew other effective remedies. Injection of botulinum toxin into aspecific muscle, commonly known as BOTOX® therapy, has been approved bythe U.S. Food and Drug Administration for treatment of cervical dystonia(an asymmetric muscular spasm in the neck that results in forcefulturning of the head), strabismus (misalignment of the eyes), focal spasmsuch as hemifacial spasm (unilateral muscle contractions of the face),and blepharospasm (involuntary forceful closure of the eyelids).Botulinum toxin also has been used to treat other conditions such as,without limitation, migraine headache, chronic low back pain, stroke,traumatic brain injury, cerebral palsy, urinary incontinence and variousdystonias. The reduction in unwanted muscle spasm afforded by botulinumtoxin therapy results in improved muscle function as well as pain reliefin treated patients. As an example, among patients treated with BOTOX®for cervical dystonia, 90% experienced improved postural deviation, and76-93% experienced pain relief following treatment.

A single BOTOX® injection generally provides therapeutic benefit for aduration of about 6 weeks to several months. Treatment is typicallyrepeated at regular intervals to obtain sustained therapeutic benefitover time. However, in some cases, patients become nonresponsive toBOTOX® therapy. Resistance to therapy can occur, for example, due to thedevelopment in the patient of antibodies that bind to and inactivate thetherapeutic toxin. Such antibody-mediated resistance to BOTOX®, whichhas been estimated to occur with a frequency of 3% to 10%, cannot bereadily distinguished from other types of BOTOX® resistance based on thesymptoms of the patient. In addition, there is currently no convenientdiagnostic test available for detecting the presence of anti-botulinumtoxin antibodies in a patient, nor is there a treatment available toprevent the onset of antibody-mediated resistance to BOTOX® therapy.

Thus, there exists a need for methods of predicting as well as reducingimmunoresistance to botulinum toxin therapy. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The present invention provides a method of predicting or determiningimmunoresistance to botulinum toxin therapy in an individual bydetermining the presence or absence in the individual of antibodiesimmunoreactive with a BoNT/A peptide having a length of at most 60 aminoacids and containing the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or a conservativevariant or immunoreactive fragment thereof, where the presence ofantibodies immunoreactive with the peptide indicates immunoresistance tobotulinum toxin therapy, with the proviso that the BoNT/A peptide is notSEQ ID NO:2.

The present invention further provides a method of preventing orreducing immunoresistance to botulinum toxin therapy in an individual byadministering to the individual a tolerogizing agent and a BoNT/Apeptide having a length of at most 60 amino acids and containing theamino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1,515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1,711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1,or 837-863 of SEQ ID NO:1, or a conservative variant or immunoreactivefragment thereof, thereby preventing or reducing immunoresistance tobotulinum toxin therapy, with the proviso that the BoNT/A peptide is notSEQ ID NO:2.

Also provided herein is a method of vaccinating an individual againstbotulinum toxin by administering to the individual a vaccine containingan adjuvant and a BoNT/A peptide which has a length of at most 60 aminoacids and contains the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or a conservativevariant or immunoreactive fragment thereof, thereby producing an immuneresponse to botulinum toxin in the individual, with the proviso that theBoNT/A peptide is not SEQ ID NO:2. In one embodiment, a method of theinvention is practiced using a BoNT/A peptide that contains the aminoacid sequence 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 ofSEQ ID NO:1, 585-611 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 739-765 ofSEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, or 823-849of SEQ ID NO:1, or a conservative variant or immunoreactive fragmentthereof, with the proviso that the BoNT/A peptide is not SEQ ID NO:2.

Also provided herein is a BoNT/A peptide that has a length of at most 60amino acids and contains the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or a conservativevariant or immunoreactive fragment thereof, with the proviso that theBoNT/A peptide is not SEQ ID NO:2.

Further provided herein is a tolerogizing composition containing atolerogizing agent and a BoNT/A peptide having a length of at most 60amino acids that includes the amino acid sequence 445-471 of SEQ IDNO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ IDNO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ IDNO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ IDNO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ IDNO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ IDNO:1823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or a conservativevariant or tolerogenic fragment thereof, with the proviso that theBoNT/A peptide is not SEQ ID NO:2.

In one embodiment, the BoNT/A peptide includes the amino acid sequence515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,585-611 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, or 823-849 of SEQ IDNO:1, or a conservative variant or tolerogenic fragment thereof, withthe proviso that the BoNT/A peptide is not SEQ ID NO:2.

The present invention further provides a vaccine composition thatcontains an adjuvant and a BoNT/A peptide having a length of at most 60amino acids and including the amino acid sequence 445-471 of SEQ IDNO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ IDNO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ IDNO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ IDNO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ IDNO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ IDNO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or aconservative variant or immunoreactive fragment thereof, with theproviso that the BoNT/A peptide is not SEQ ID NO:2. In one embodiment,the BoNT/A peptide includes the amino acid sequence 515-541 of SEQ IDNO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 585-611 of SEQ IDNO:1, 655-681 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 781-807 of SEQ IDNO:1, 809-835 of SEQ ID NO:1, or 823-849 of SEQ ID NO:1, or aconservative variant or immunoreactive fragment thereof, with theproviso that the BoNT/A peptide is not SEQ ID NO:2.

The invention additionally provides a method of preparing an anti-BoNT/Aantibody by administering to an animal a BoNT/A peptide having a lengthof at most 60 amino acids and containing the amino acid sequence 445-471of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or aconservative variant or immunoreactive fragment thereof; collecting fromthe animal a sample containing an antibody or antibody-producing cell;and processing the sample to isolate the anti-BoNT/A antibody, with theproviso that the BoNT/A peptide is not SEQ ID NO:2. In one embodiment, amethod of the invention is practiced with a BoNT/A peptide containingthe amino acid sequence 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 655-681 of SEQ ID NO:1,739-765 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,or 823-849 of SEQ ID NO:1, or a conservative variant or immunoreactivefragment thereof, with the proviso that the BoNT/A peptide is not SEQ IDNO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows synthetic consecutive overlapping peptides of the H_(N)domain of BoNT/A having the indicated residues of SEQ ID NO:1. Regionsof overlap with adjacent peptides are underlined and bolded. FIG. 1Bshows synthetic consecutive overlapping peptides of the H_(C) domain ofBoNT/A having the indicated residues of SEQ ID NO:1. Regions of overlapwith adjacent peptides are underlined and bolded. The L-peptide controlsequence is shown as SEQ ID NO:1.

FIG. 2 shows binding of human anti-pentavalent botulinum toxoidantibodies to overlapping synthetic peptides spanning the BoNT/A H_(N)domain and to active H_(C) peptides. Also shown are binding to L-Peptideand full-length BoNT/A as negative and positive controls, respectively.

FIG. 3 shows binding of anti-pentavalent botulinum toxoid antibodies ofICR outbred mice to synthetic overlapping peptides spanning the BoNT/AH_(N) domain. Also shown are binding to L-Peptide and full-length BoNT/Aas negative and positive controls, respectively.

FIG. 4 shows binding of chicken anti-BoNT/A antibodies to 60 syntheticoverlapping peptides spanning the entire H-subunit of BoNT/A. Also shownare binding to L-Peptide and full-length BoNT/A as negative and positivecontrols, respectively.

FIG. 5 shows binding of horse anti-BoNT/A antibodies to active BoNT/Aoverlapping synthetic peptides spanning the BoNT/A H_(N) domain and toactive H_(C) peptides. Also shown are binding to L-Peptide andfull-length BoNT/A as negative and positive controls, respectively.

FIG. 6 shows amino acid sequences of the HN domain of BoNT/A (SEQ IDNO:1); the Hc domain of BoNT/A (SEQ ID NO:1); the L peptide (SEQ IDNO:1); and amino acids 731 to 787 of BoNT/E (SEQ ID NO:4).

FIG. 7 shows proliferative responses of LNC (8×10⁵ cells/well) fromBALB/c mice primed with 1:g of BoNT/A toxoid to BoNT/A, BoNT/B and TeNT.

FIG. 8 shows proliferative responses of LNC (5×10⁵ cells/well) of Balb/cmice after 1 injection or after 3 injections with BoNT/A toxoid(1:g/mouse/injection).

FIG. 9 shows proliferative responses of BoNT/A, BoNT/B and TeNT of LNC(7×10⁵ cells/well) from SJL mice primed with 1:g BoNT/A toxoid.

FIG. 10 shows proliferative responses of LNC (5×10⁵ cells/well) of SJLmice to various synthetic BoNT/A peptides after 1 injection or after 3injections with BoNT/A toxoid (1: g/mouse/injection).

FIG. 11 shows binding of Balb/c anti-BoNT/A antibodies to BoNT/A and tooverlapping synthetic peptides spanning the H_(N)-domain. Antisera wereassayed at two dilutions (1:500 and 1:250, vol/vol).

FIG. 12 shows binding of SJL anti-BoNT/A antibodies to BoNT/A and tooverlapping synthetic peptides of the H_(N)-chain. Antisera were assayedat dilutions of 1:500 and 1:250.

FIG. 13 shows a comparison of the binding profiles of BALB/c and SJLanti-BoNT/A toxoid antibodies at an antisera dilution of 1:250(vol/vol), to BoNT/A and to overlapping synthetic peptides of theH_(N)-domain.

FIG. 14 shows protective activity of different dilutions of BALB/c andSJL anti-BoNT/A antisera. The results are expressed in percent survivalto BoNT/A challenge versus antiserum dilution.

FIG. 15 shows protective activity of BALB/c and SJL anti-BoNT/A antiseraobtained on day 36 after a first immunization. Antisera of each strainwere tested at the indicated dilutions for their ability to protectrecipient ICR mice against 1.05×LD₁₀₀ of active BoNT/A. The results areexpressed in percent survival to BoNT/A challenge versus antiserumdilution.

FIG. 16 shows binding of BALB/c total antibodies in non-protecting (day26) and protecting (day 36) anti-BoNT/A antisera to the overlappingsynthetic peptides spanning the entire H chain and to the L-peptidearound the enzyme active site of the L chain of BoNT/A. Results are fromtriplicate analyses and are expressed in net cpm, after correction fornonspecific binding in control wells coated with unrelated protein (BSA)or peptides and also controls of bound label to BoNT/A and to peptidesin pre-immune serum of the same mice.

FIG. 17 shows binding of SJL total antibodies in non-protecting (day 26)and protecting (day 36) anti-BoNT/A antisera to the overlappingsynthetic peptides spanning the entire H chain and to the L-peptidearound the enzyme active site of the L chain of BoNT/A. Results are fromtriplicate analyses and are expressed in net cpm, after correction asdescribed above.

FIG. 18 shows binding of BALB/c IgG antibodies in non-protecting (day26) and protecting (day 36) anti-BoNT/A antisera to the overlappingsynthetic peptides spanning the entire H chain and to the L-peptidearound the enzyme active site of the L chain of BoNT/A. Results are fromtriplicate analyses and are expressed in net cpm, after correction asdescribed above.

FIG. 19 shows binding of SJL IgG antibodies in non-protecting (day 26)and protecting (day 36) anti-BoNT/A antisera to the overlappingsynthetic peptides spanning the entire H chain and to the L-peptidearound the enzyme active site of the L chain of BoNT/A. Results are fromtriplicate analyses and are expressed in net cpm, after correction asdescribed above.

FIG. 20 shows a comparison of IgG antibody binding profiles fromprotective (day 36) BALB/c and SJL antisera. The data are the same asthose shown in FIGS. 4 and 5. Binding studies were performed withantisera at a dilution of 1:250 (vol/vol).

FIG. 21 shows binding to BoNT/A of antibodies in sera from CD patients(n=28) that are MPA-positive for anti-BoNT/A antibodies and in normalcontrols (n=10). Results are average of three experiments expressed inratios of antibodies bound to BoNT/A over antibodies bound to negativecontrols.

FIG. 22 shows binding to BoNT/B of antibodies in MPA anti-BoNT/Apositive sera from CD patients (n=28) and in normal controls (n=10).Results are in ratios of antibodies bound to BoNT/B over antibodiesbound to negative controls.

FIG. 23 shows mapping of the antibody recognition profile in serumsamples from 13 CD patients. Results are expressed as a ratio ofantibodies bound to peptides in the CD sera/average of antibodies boundby four negative control peptides.

FIG. 24 shows mapping of the antibody recognition profile in serumsamples from 15 CD patients. Results are expressed as a ratio ofantibodies bound to peptides in the CD sera/average of antibodies boundby four negative control peptides.

FIG. 25 shows mapping of the antibody recognition profile in serumsamples from 28 CD patients. Results are expressed as a ratio ofantibodies bound to peptides in the CD sera/average of antibodies boundby four negative control peptides.

FIG. 26 shows binding to peptide N25 of antibodies in MPA-positive serafrom CD patients (n=28) and in normal controls (n=10). Results are theaverage of four experiments and are expressed as a ratio of (antibodiesbound to peptide N25)/(average of antibodies bound by negative controlpeptides N2, N12, C17 and C23).

FIG. 27 shows binding to peptide C10 of antibodies in MPA-positive serafrom CD patients (n=28) and in normal controls (n=10). Results are theaverage of four experiments and are expressed as a ratio of (antibodiesbound to peptide C10)/(average of antibodies bound by negative controlpeptides N2, N12, C17 and C23).

FIG. 28 shows binding to peptide C15 of antibodies in MPA-positive serafrom CD patients (n=28) and in normal controls (n=10). Results are theaverage of four experiments and are expressed as a ratio of (antibodiesbound to peptide C10)/(average of antibodies bound by negative controlpeptides N2, N12, C17 and C23).

FIG. 29 shows binding to peptide C31 of antibodies in MPA-positive serafrom CD patients (n=28) and in normal controls (n=10). Results are theaverage of four experiments and are expressed as a ratio of (antibodiesbound to peptide C10)/(average of antibodies bound by negative controlpeptides N2, N12, C17 and C23).

FIG. 30 shows binding to peptides (N25+C10) of antibodies inMPA-positive sera from CD patients (n=28) and in normal controls (n=10).The results, which are the average of four experiments, are expressed asa ratio of (antibodies bound to peptide N25+C10)/(average of antibodiesbound by negative control peptides N2, N12, C17 and C23).

FIG. 31 shows binding peptides (N25+C10+C31) of antibodies inMPA-positive sera from CD patients (n=28) and in normal controls (n=10).Results, which are the average of four experiments, are expressed as aratio of (antibodies bound to peptides N25+C10+C31)/(average ofantibodies bound by negative control peptides N2, N12, C17 and C23).

FIG. 32 shows binding to peptides (N25+C10+C15) of antibodies inMPA-positive sera from CD patients (n=28) and in normal controls (n=10).Results, which are the average of four experiments, are expressed as aratio of (antibodies bound to peptides N25+C10+C15)/(average ofantibodies bound by negative control peptides N2, N12, C17 and C23).

FIG. 33 shows binding to peptides (N25+C10+C15+C31) of antibodies inMPA-positive sera from CD patients (n=28) and in normal controls (n=10).Results, which are the average of four experiments, are expressed as aratio of (antibodies bound to peptides N25+C10+C15+C31)/(average ofantibodies bound by negative control peptides N2, N12, C17 and C23).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to botulinum neurotoxin A (BoNT/A) peptides thatrepresent the complete repertoire of epitopes from the H_(N) domain ofBoNT/A recognized by antibodies from humans immunized with pentavalentbotulinum toxoid. BoNT/A peptides of the invention, and antibodies thatbind to such peptides, are useful, for example, in methods forpredicting or diagnosing immunoresistance to botulinum toxin therapy,for reducing the development of such immunoresistance, and for boostingimmunity against unwanted botulinum toxicity.

Botulinum neurotoxins (BoNTs) are a group of protein neurotoxinsproduced by Clostridium botulinum that are among the most toxicsubstances known to man. Seven immunologically distinct BoNT serotypes(A through G) are known, including two subtypes of type C(C1 and C2).Botulinum neurotoxins are synthesized from a single polypeptide chainwith a molecular weight of about 150 KDa, which is activated aftersecretion by nicking of a single peptide bond by an endogenous orexogenous protease. In C. botulinum strains that produce BoNTs A, C, D,and some types of B and F, the proteolytic enzyme is endogenous, whilein other strains such as those that produce type E and some types B andF, the proteolytic enzyme is exogenous. The nicking of the progenitortoxin generally results in generation of two subunits, a 100 KDa heavychain (H chain) and a 50 KDa light chain (L chain). With the exceptionof BoNT/C2, the two subunits are held together by a disulfide bond,which is important for neurotoxicity of toxin added extracellularly.

The cell intoxication mechanism of the BoNTs consists of four distinctsteps: (1) binding; (2) internalization; (3) membrane translocation; and(4) enzymatic target modification. The carboxy-terminal part of theheavy chain (denoted the “H_(C)” domain) functions in neurospecificbinding, while the amino-terminal portion of the H chain (denoted the“H_(N)” domain) functions in membrane translocation. The L chain is azinc proteinase responsible for the intracellular activity of BoNTs, andhas specificity for one or more of proteins involved in releasingacetylcholine into the neuromuscular junction.

The complete primary structures of BoNTs A through G have beendetermined (Binz et al., J. Biol. Chem. 265:9153 (1990); Willems et al.,Res. Microbiol. 144:547 (1993); Hutson et al., Curr. Microbiol. 28:101(1994); Campbell et al., Clin. Microbiol. 31:2255 (1993); Hauser et al.,Nucl. Acids Res. 18:4924 (1990); Hauser et al., Bacteriol. 175:7260(1993); Kimura et al., Biochem. Biophys. Res. Commun. 171:1304 (1990);Kimura et al., Appl. Environ. Microbiol. 57:1168 (1991); Hauser et al.,Toxicon 33:515 (1995); Binz et al., Nucl. Acids Res. 18:5556 (1990);Sunagawa et al., J. Vet. Med. Sci. 54:905 (1992); Campbell et al., J.Clin. Microbiol. 31:2255 (1993); Poulet et al., Biochem. Biophys.Research Commun. 183:107 (1992); Whelan et al., Eur. J. Biochem. 204:657(1992); Campbell et al., J. Clin. Microbiol. 31:2255 (1993); East etal., FEMS Microbiol. Lett. 75:225 (1992); and Campbell et al., Biochim.Biophys. Acta 1216:487 (1993)). In addition, the disulfide pairing inBoNT/A has been determined. Several regions of homology exist within theamino acid sequences of the different serotypes of BoNT, as described inAtassi et al., Critical Reviews in Immunology 19:219-260 (1999).

The present invention relates to the discovery of small BoNT/A peptideswhich elicit antibody responses and represent the repertoire of epitopeswithin the BoNT/A H_(N) domain recognized by four animal species,including humans. As shown herein in Examples I through IV, antigenicregions of the BoNT/A H_(N) domain were mapped using human, mouse,chicken and horse sera obtained following immunization with BoNT/A.Mapping was performed using twenty nine BoNT/A peptides, each containingnineteen residues, that overlap consecutively by five residues andcorrespond to the entire length of the H_(N) domain. The amino acidsequences of peptides used for mapping are shown in FIG. 1A. Resultsfrom the mapping studies revealed eighteen segments of BoNT/A thatrepresent the complete repertoire of continuous antigenic regions on theBoNT/A H_(N) domain.

As disclosed herein in Example VII, T- and B-cell recognition profilesof the BoNT/A H_(N) domain were mapped in two inbred mouse strains,BALB/c (H-2^(d)) and SJL (H-2^(s)), that are high responders to BoNT/A.As summarized in Table 5, the results obtained with the twohigh-responder mouse strains demonstrate that responses to each antibodyand T cell epitope are under separate genetic control and furtherindicate that there is partial overlap between antibody and T cell H_(N)recognition regions.

Resistance in the majority of patients is associated with the appearanceof blocking anti-toxin antibodies in patient serum (Goschel et al., Exp.Neurol. 147:96-102 (1997); Atassi and Oshima, Crit. Revs. Immunol.19:219-260 (1999); Jankovic, in Brian et al., Eds., Scientific andTherapeutic Aspects of Botulinum Toxin, pp. 409-415, Lippincott Williams& Wilkins; Philadelphia, Pa. (2002). While all patient antibodyresponses against the toxin are not observed initially, additionalinjections of toxin appear to cause a switch of the non-blockingantibodies in the patient's serum to blocking antibodies. As furtherdisclosed herein in Example VIII, the epitope recognition profile wascompared in inbred BALB/c and SJL mice before and after the switch fromproduction of non-protective to protective antibodies. The resultsdisclosed herein demonstrated only slight differences in the epitoperecognition profiles of non-protective and protective antisera,indicating that changes in antibody binding may not always protection,or lack thereof, by serum from a given strain (FIGS. 16 and 17).Furthermore, as shown in FIGS. 18 and 19, IgG antibodies in theprotective antisera of each mouse strain bound to the same peptides asdid total antibodies (IgG and IgM) in the same serum, while in bothmouse strains, non-protective antisera contained few, if any, IgGantibodies to these peptides. These results appear to indicate thatprotection can be a function of immunoglobulin class, with IgGantibodies conferring protection against botulinum toxin.

Additional studies disclosed herein in Example IX demonstrate that invitro binding assays performed in the presence of excess tetanus toxoidcan be used to determine the levels of blocking or protectiveanti-BoNT/A antibodies in human serum samples. In particular, sera from28 cervical dystonia patients containing protective antibodies asindicated by the mouse protection assay (MPA) and 10 negative controlhuman sera from unimmunized controls were analyzed. As shown in FIGS. 24to 26 and summarized in Table 6, peptides which bound antibodies inMPA-positive human patient sera also bound antibodies in hyperimmunemouse sera, while the antibody-binding profile of patient sera was morerestricted than the profile of the hyperimmune sera. As furtherdisclosed herein in Example IX, several peptides bound antibodies inmost patient samples, with 25 out of 28 sera containing antibodies thatbound peptide N25; 24 out of 28 sera containing antibodies that boundpeptide C10; and lower binding to peptides C15, C20 and C31 seen in themajority of patient samples. These results indicate that, while there issome variability among the peptide-binding profiles of MPA-positivehuman sera, several synthetic BoNT/A peptides bind antibodies in thelarge majority of human patient sera that contain protective antibodies.

Further results disclosed herein demonstrate that an assay based on acombination of two or more synthetic BoNT/A peptides can be useful fordetecting the presence of protective or blocking antibodies in the seraof patients treated with a BoNT/A formulation. As shown in FIG. 30, inan assay combining synthetic peptides N25 and C10, 25 out of 28 (89.3%)of the MPA-positive CD sera were discriminated from control sera. FIG.32 shows that a combination of the synthetic peptides N25, C10 and C15also served to distinguish 25 out of 28 (89.3%) of the MPA-positive CDsera from controls. Thus, the results disclosed herein demonstrate thata combination assay using peptides N25 and C10, or peptides N25, C10 andC15 can be useful for detecting the presence of specific anti-toxinantibodies in BOTOX® treated patients. Furthermore, one or a combinationof the synthetic peptides N25, C10, N15, N20 or N31, or a conservativevariant or immunoreactive fragment thereof, also can be useful in avariety of diagnostic or therapeutic applications including, withoutlimitation, methods of predicting or determining immunoresistance tobotulinum toxin therapy; methods of preventing or reducingimmunoresistance to botulinum toxin therapy and related tolerogeniccompositions; methods of vaccinating against botulinum toxin and relatedvaccine compositions; methods of removing anti-botulinum toxinantibodies from blood, plasma or serum and affinity-matrices usefultherefore; and new therapeutic formulations for blocking the effect ofneutralizing antibodies in situ. Such therapeutic formulations includeexcess synthetic protective antibody-binding peptides together with theactive toxin formulation.

BoNT/A Peptides

The present invention provides a BoNT/A peptide that has a length of atmost 60 amino acids and contains the amino acid sequence 445-471 of SEQID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or aconservative variant or immunoreactive fragment thereof, with theproviso that the BoNT/A peptide is not SEQ ID NO:2.

In one embodiment, such a BoNT/A peptide includes the amino acidsequence 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQID NO:1, 585-611 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 739-765 of SEQID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, or 823-849 ofSEQ ID NO:1, or a conservative variant or immunoreactive fragmentthereof, with the proviso that the BoNT/A peptide is not SEQ ID NO:2. ABoNT/A peptide of the invention can have, for example, a length of atmost 40 amino acids or a length of at most 25 amino acids.

In another embodiment, a BoNT/A peptide of the invention has a length ofat most 60 amino acids and includes the amino acid sequence 445-471 ofSEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 ofSEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 ofSEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 ofSEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 ofSEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 ofSEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or aconservative variant thereof. In a further embodiment, a BoNT/A peptideof the invention has a length of at most 60 amino acids and includes theamino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1,515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1,711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1,or 837-863 of SEQ ID NO:1, or an immunoreactive fragment thereof, withthe proviso that the BoNT/A peptide is not SEQ ID NO:2. In a furtherembodiment, a BoNT/A peptide of the invention has a length of at most 60amino acids and includes the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1. In still a furtherembodiment, a BoNT/A peptide of the invention has the amino acidsequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 ofSEQ ID NO:1.

As used herein, the term “peptide” means two or more amino acidscovalently bonded together. The term “BoNT/A peptide,” as used herein,means a peptide having a length of at most 60 amino acids which includesan amino acid sequence having at least 50% amino acid identity with aportion of the BoNT/A sequence SEQ ID NO:1. Thus, a BoNT/A peptide canbe, for example, a peptide of at most 60 amino acids having an aminoacid sequence corresponding to a portion of the naturally occurringBoNT/A sequence SEQ ID NO:1, a peptide having one or more conservativeor non-conservative substitutions relative to a portion of SEQ ID NO:1,a conservative variant of a portion of the BoNT/A sequence SEQ ID NO:1,or an immunoreactive fragment. The term “BoNT/A peptide” encompasses“variants,” “conservative variants” and “immunoreactive fragments,” eachof which is described further below. Specifically excluded from thedefinition of a BoNT/A peptide is the 57-mer SEQ ID NO:2 described inKubota et al., Applied Envir. Microbiol. 63:1214-1218 (1997). In oneembodiment, a BoNT/A peptide is not SEQ ID NO:10 or a fragment thereof.

A BoNT/A peptide can have any of a variety of lengths up to 60 aminoacids. In particular embodiments, a BoNT/A peptide includes at most 55amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 aminoacids, 30 amino acids or 25 amino acids. In further embodiments, aBoNT/A peptide of the invention includes at most 55 amino acids of SEQID NO:1, at most 50 amino acids of SEQ ID NO:1, at most 45 amino acidsof SEQ ID NO:1, at most 40 amino acids of SEQ ID NO:1, at most 35 aminoacids of SEQ ID NO:1, at most 30 amino acids of SEQ ID NO:1, at most 25amino acids of SEQ ID NO:1, at most 20 amino acids of SEQ ID NO:1 or atmost 15 amino acids of SEQ ID NO:1 and further includes at least one ofthe following BoNT/A amino acid sequences: amino acids 445-471 of SEQ IDNO:1, amino acids 487-513 of SEQ ID NO:1, amino acids 515-541 of SEQ IDNO:1, amino acids 529-555 of SEQ ID NO:1, amino acids 543-569 of SEQ IDNO:1, amino acids 557-583 of SEQ ID NO:1, amino acids 585-611 of SEQ IDNO:1, amino acids 599-625 of SEQ ID NO:1, amino acids 627-653 of SEQ IDNO:1, amino acids 655-681 of SEQ ID NO:1, amino acids 669-695 of SEQ IDNO:1, 683-709 of SEQ ID NO:1, amino acids 711-737 of SEQ ID NO:1, aminoacids 739-765 of SEQ ID NO:1, amino acids 767-793 of SEQ ID NO:1, aminoacids 781-807 of SEQ ID NO:1, amino acids 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or amino acids 837-863 of SEQ ID NO:1 or aconservative variant or immunoreactive fragment thereof.

Conservative Variants

A BoNT/A peptide can contain conservative amino acid substitutions thatdo not substantially alter the antigenicity of the reference BoNT/Apeptide having a sequence corresponding to a portion of SEQ ID NO:1.Such a “conservative variant” can function in substantially the samemanner as a BoNT/A reference peptide, and can be substituted for thereference peptide in a method of the invention. As used herein inreference to a specified amino acid sequence, the term “conservativevariant” means a sequence in which a first amino acid is replaced byanother amino acid or amino acid analog that has least one biochemicalproperty similar to that of the first amino acid; similar propertiesinclude, without limitation, similar size, charge, hydrophobicity orhydrogen-bonding capacity or a combination thereof. It is understoodthat conservative variants of BoNT/A peptides encompass, for example,conservative variants containing one, two, three, four or more aminoacid substitutions relative to a portion of SEQ ID NO:1 and that suchvariants can include naturally and non-naturally occurring amino acidanalogs as described further below.

As a non-limiting example, a conservative variant can be a sequence inwhich a first uncharged polar amino acid is conservatively substitutedwith a second (non-identical) uncharged polar amino acid such ascysteine, serine, threonine, tyrosine, glycine, glutamine or asparagineor an analog thereof. A conservative variant also can be, for example, asequence in which a first basic amino acid is conservatively substitutedwith a second basic amino acid such as arginine, lysine, histidine,5-hydroxylysine, N-methyllysine or an analog thereof. Similarly, aconservative variant can be, for example, a sequence in which a firsthydrophobic amino acid is conservatively substituted with a secondhydrophobic amino acid such as alanine, valine, leucine, isoleucine,proline, methionine, phenylalanine or tryptophan or an analog thereof.In the same way, a conservative variant can be, for example, a sequencein which a first acidic amino acid is conservatively substituted with asecond acidic amino acid such as aspartic acid or glutamic acid or ananalog thereof; a sequence in which an aromatic amino acid such asphenylalanine is conservatively substituted with a second aromatic aminoacid or amino acid analog, for example, tyrosine; or a sequence in whicha first relatively small amino acid such as alanine is substituted witha second relatively small amino acid or amino acid analog such asglycine or valine or an analog thereof.

As a non-limiting example, conservative variants of BoNT/A peptidesinclude conservative variants of a BoNT/A peptide having residues445-471 of SEQ ID NO:1; such conservative variants can have, forexample, an arginine for lysine substitution at position 456 and anisoleucine for leucine substitution at position 462. Additionalconservative variants include conservative variants of the BoNT/Apeptide having residues 487-513 of SEQ ID NO:1; such conservativevariants can have, for example, a glutamic acid for aspartic acidsubstitution at position 497; an asparagine for glutamine substitutionat position 500; and a phenylalanine for tyrosine substitution atposition 502.

Immunoreactive Fragments

The present invention further provides immunoreactive fragments of theBoNT/A peptides of the invention. Such immunoreactive fragments can besubstituted for the corresponding full-length BoNT/A peptide in a methodof the invention. As used herein in reference to a specified amino acidsequence, the term “immunoreactive fragment” means a portion of thespecified BoNT/A amino acid sequence, or a conservative variant thereof,capable of selective antibody binding. An immunoreactive fragment can becapable of selective antibody binding to anti-BoNT/A antibodies from oneor more species. In one embodiment, an immunoreactive fragment bindsanti-BoNT/A antibodies from human sera.

An immunoreactive fragment of a BoNT/A peptide generally has from aboutsix amino acids to 60 amino acids. An immunoreactive fragment of aBoNT/A peptide can have, for example, a length of at least 5, 6, 7, 8,9, 10, 12, 15, 18, 20 or 25 amino acids. An immunoreactive fragment of aBoNT/A peptide also can have, for example, a length of at most 8, 9, 10,12, 15, 18, 20, 25, 30 or 35 amino acids. In particular embodiments, animmunoreactive fragment of a BoNT/A peptide has from five to fifty aminoacids, from eight to fifty amino acids, from ten to fifty amino acids,from five to twenty amino acids, from eight to twenty amino acids, fromten to twenty amino acids, from twelve to twenty amino acids or fromfifteen to twenty amino acids. An immunoreactive fragment can have anynumber of conservative substitutions as discussed above.

An immunoreactive fragment can be identified using any of a variety ofroutine assays for detecting peptide antigen-antibody complexes, thepresence of which is an indicator of selective binding. Such assaysinclude, without limitation, enzyme-linked immunosorbent assays,radioimmunoassays, western blotting, enzyme immunoassays, fluorescenceimmunoassays, luminescent immunoassays and the like and generally areequivalent to the radioimmunoassay disclosed herein in Example I.Methods for detecting a complex between a peptide and an antibody, andthereby determining if the peptide is an “immunoreactive fragment” arewell known to those skilled in the art and are described, for example,in Harlow and Lane, Using Antibodies: A Laboratory Manual New York: ColdSpring Harbor Laboratory Press (1998).

Variants

A BoNT/A peptide useful in the compositions and methods of the inventionalso can be a BoNT/A “variant,” which as defined herein has an aminoacid sequence having at least 50% amino acid identity with a portion ofSEQ ID NO:1 and is capable of selective antibody binding. In particularembodiments, a BoNT/A variant has an amino acid sequence with at least65% amino acid identity to a portion of SEQ ID NO:1 having at least 10contiguous residues, 15 contiguous residues, 20 contiguous residues, or25 contiguous residues of SEQ ID NO:1. In further embodiments, a BoNT/Avariant has an amino acid sequence with at least 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to aportion of SEQ ID NO:1 having at least 10 contiguous residues, 15contiguous residues, 20 contiguous residues, or 25 contiguous residuesof SEQ ID NO:1.

Peptidomimetics

A BoNT/A peptide such as, for example, a conservative variant,immunoreactive fragment or variant, also can contain one or morenon-amide linkages between amino acids, or one or more amino acidanalogs or mimetics, and further can have, for example, a cyclic orother conformationally constrained structure. As used herein, the term“amino acid” is intended to mean both naturally occurring andnon-naturally occurring amino acids as well as amino acid analogs andmimetics. Naturally occurring amino acids include the 20 (L)-amino acidsutilized during protein biosynthesis as well as others such as, withoutlimitation, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,homocysteine, citrulline and ornithine. Non-naturally occurring aminoacids include, but are not limited to, (D)-amino acids, norleucine,norvaline, p-fluorophenylalanine, ethionine and the like. Amino acidanalogs include modified forms of naturally and non-naturally occurringamino acids. Such modifications can include, for example, substitutionor replacement of chemical groups or moieties on the amino acid or byderivatization of the amino acid. Amino acid mimetics include, forexample, organic structures that exhibit functionally similar propertiessuch as charge and charge spacing characteristic of the reference aminoacid. As an example, an organic structure that mimics arginine can havea positive charge moiety located in similar molecular space and havingthe same degree of mobility as the e-amino group of the side chain ofthe naturally occurring arginine amino acid. Mimetics also includeconstrained structures which maintain advantageous spacing or chargeinteractions of the amino acid or amino acid functional groups. Oneskilled in the art understands that these and other well known aminoacid analogs and mimetics can be useful in the BoNT/A peptides of theinvention.

Specific examples of amino acid analogs and mimetics can be founddescribed in, for example, Roberts and Vellaccio, The Peptides:Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341,Academic Press, Inc., New York, N.Y. (1983). Other non-limiting examplesinclude peralkylated amino acids, particularly permethylated aminoacids. See, for example, Combinatorial Chemistry, Eds. Wilson andCzarnik, Ch. 11, p. 235, John Wiley & Sons Inc., New York, N.Y. (1997).Further non-limiting examples include amino acids in which the amideportion has been replaced, for example, by a sugar ring, steroid,benzodiazepine or carbo cycle. See, for instance, Burger's MedicinalChemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620,John Wiley & Sons Inc., New York, N.Y. (1995). Methods for synthesizingpeptides include well known chemical synthesis methods. See, forexample, U.S. Pat. No. 5,420,109; Chapter 7 of Bodanzsky, Principles ofPeptide Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York,N.Y. (1984 & 1993); and Stewart and Young, Solid Phase PeptideSynthesis, (2d ed.), Pierce Chemical Co., Rockford, Ill. (1984).

Heterologous Fusions

A peptide of the invention can be fused to a heterologous protein, whichis a protein derived from a source other than the gene encoding thepeptide of the invention, to form a chimeric BoNT/A protein. Such achimeric BoNT/A protein of the invention can have a variety of lengthsincluding, but not limited to, a length of at most 100 residues, at most200 residues, at most 300 residues, at most 400 residues, at most 500residues, at most 800 residues or at most 1000 residues. Non-limitingexamples of chimeric BoNT/A proteins include fusions of BoNT/A peptideswith immunogenic polypeptides, such as flagellin and choleraenterotoxin; fusions of BoNT/A peptides with immunomodulatorypolypeptides, such as IL-2 and B7-1; fusions of BoNT/A peptides withtolerogenic polypeptides, such as another BoNT/A peptide and an antibodyselectively reactive with interleukin-12; and fusions of BoNT/A peptideswith synthetic sequences.

Methods of Predicting or Determining Immunoresistance

BoNT/A peptides, including conservative variants and immunoreactivefragments of the amino acid sequences disclosed herein, each contain oneor more epitopes recognized by antibodies contained in antisera fromanimals, for example, humans, immunized with BoNT/A. As described above,patients treated with botulinum toxin can develop immunoresistance tothe therapeutic toxin, reducing or eliminating the beneficial effect ofbotulinum toxin therapy. The BoNT/A peptides of the invention are usefulin methods of predicting or determining immunoresistance to botulinumtoxin therapy in an individual.

Thus, the present invention provides a method of predicting ordetermining immunoresistance to botulinum toxin therapy in an individualby determining the presence or absence in the individual of antibodiesimmunoreactive with a BoNT/A peptide having a length of at most 60 aminoacids and containing the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or a conservativevariant or immunoreactive fragment thereof, where the presence ofantibodies immunoreactive with the peptide indicates immunoresistance tobotulinum toxin therapy, with the proviso that the BoNT/A peptide is notSEQ ID NO:2.

In a method of the invention, the BoNT/A peptide can include, forexample, the amino acid sequence 515-541 of SEQ ID NO:1, 529-555 of SEQID NO:1, 543-569 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 655-681 of SEQID NO:1, 739-765 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQID NO:1, or 823-849 of SEQ ID NO:1, or a conservative variant orimmunoreactive fragment thereof, with the proviso that the BoNT/Apeptide is not SEQ ID NO:2. A BoNT/A peptide useful in the invention canhave, for example, a length of at most 40 amino acids or a length of atmost 25 amino acids.

In one embodiment, the BoNT/A peptide includes the amino acid sequence445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1,529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1,585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1,669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1,739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1,809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ IDNO:1, or a conservative variant thereof. In another embodiment, theBoNT/A peptide includes the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or an immunoreactivefragment thereof, with the proviso that the BoNT/A peptide is not SEQ IDNO:2. In a further embodiment, the BoNT/A peptide includes the aminoacid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 ofSEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 ofSEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 ofSEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 ofSEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 ofSEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863of SEQ ID NO:1. In still a further embodiment, the BoNT/A peptide hasthe amino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1,515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1,711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1,or 837-863 of SEQ ID NO:1.

A method of the invention for predicting or determining immunoresistanceto botulinum toxin therapy can optionally include determining thepresence or absence of antibodies immunoreactive with a H_(C) peptide.H_(C) peptides useful in the invention include, without limitation,those including the amino acid sequence 855-873 of SEQ ID NO:1, 869-887of SEQ ID NO:1, 883-901 of SEQ ID NO:1, 897-915 of SEQ ID NO:1, 911-929of SEQ ID NO:1, 925-943 of SEQ ID NO:1, 939-957 of SEQ ID NO:1, 953-971of SEQ ID NO:1, 967-985 of SEQ ID NO:1, 981-999 of SEQ ID NO:1, 995-1013of SEQ ID NO:1, 1009-1027 of SEQ ID NO:1, 1023-1041 of SEQ ID NO:1,1037-1055 of SEQ ID NO:1, 1051-1069 of SEQ ID NO:1, 1065-1083 of SEQ IDNO:1, 1079-1097 of SEQ ID NO:1, 1093-1111 of SEQ ID NO:1, 1107-1125 ofSEQ ID NO:1, 1121-1139 of SEQ ID NO:1, 1135-1153 of SEQ ID NO:1,1149-1167 of SEQ ID NO:1, 1163-1181 of SEQ ID NO:1, 1177-1195 of SEQ IDNO:1, 1191-1209 of SEQ ID NO:1, 1205-1223 of SEQ ID NO:1, 1219-1237 ofSEQ ID NO:1, 1233-1251 of SEQ ID NO:1, 1247-1265 of SEQ ID NO:1,1261-1279 of SEQ ID NO:1, or 1275-1296 of SEQ ID NO:1, or a conservativevariant or immunoreactive fragment thereof. In one embodiment, the Hcpeptide contains the amino acid sequence 939-957 of SEQ ID NO:1, 953-971of SEQ ID NO:1, 967-985 of SEQ ID NO:1, 981-999 of SEQ ID NO:1, 995-1013of SEQ ID NO:1, 1009-1027 of SEQ ID NO:1, 1023-1041 of SEQ ID NO:1,1037-1055 of SEQ ID NO:1, 1051-1069 of SEQ ID NO:1, 1065-1083 of SEQ IDNO:1, 1079-1097 of SEQ ID NO:1, 1093-1111 of SEQ ID NO:1, 1107-1125 ofSEQ ID NO:1, 1121-1139 of SEQ ID NO:1, 1135-1153 of SEQ ID NO:1,1149-1167 of SEQ ID NO:1, 1163-1181 of SEQ ID NO:1, 1177-1195 of SEQ IDNO:1, 1191-1209 of SEQ ID NO:1, 1205-1223 of SEQ ID NO:1, 1219-1237 ofSEQ ID NO:1, 1233-1251 of SEQ ID NO:1, 1247-1265 of SEQ ID NO:1,1261-1279 of SEQ ID NO:1, or 1275-1296 of SEQ ID NO:1, or animmunoreactive fragment thereof.

In one embodiment, a method of the invention involves determining thepresence or absence of antibodies immunoreactive with two or more BoNT/Apeptides. In another embodiment, a method of the invention involvesdetermining the presence or absence of antibodies immunoreactive withfive or more BoNT/A peptides. In a further embodiment, a method of theinvention involves determining the presence or absence of antibodiesimmunoreactive with ten or more BoNT/A peptides. The two or more, fiveor more, or ten or more BoNT/A peptides can be optionally immobilized ona solid support.

The present invention also provides methods of predicting or determiningimmunoresistance to botulinum toxin therapy in an individual bydetermining the presence or absence in the individual of antibodiesimmunoreactive with two or more of the following amino acid sequences:residues 785-803 of SEQ ID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10];1051-1069 of SEQ ID NO: 1 [C15]; 1121-1139 of SEQ ID NO: 1 [C20]; and1275-1296 of SEQ ID NO: 1 [C31], or a conservative variant orimmunoreactive fragment of any of these sequences, where the presence ofantibodies immunoreactive with the two or more amino acid sequencesindicates immunoresistance to botulinum toxin therapy. In oneembodiment, one of the amino acid sequence includes residues 785-803 ofSEQ ID NO: 1 [N25] or a conservative variant or immunoreactive fragmentof this sequence. In another embodiment, a method of the invention ispracticed by determining the presence or absence in the individual ofantibodies immunoreactive with the following two amino acid sequences:785-803 of SEQ ID NO: 1 [N25]; and 981-999 of SEQ ID NO: 1 [C10], or aconservative variant or immunoreactive fragment of any of thesesequences. In still a further embodiment, a method of the invention ispracticed by determining the presence or absence in the individual ofantibodies immunoreactive with the following three amino acid sequences:785-803 of SEQ ID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10]; and1051-1069 of SEQ ID NO: 1 [C15], or a conservative variant or animmunoreactive fragment of any of these sequences. It is understood thatthe two or more amino acid sequences can be provided separately or aspart of a compound molecule such as a chimeric synthetic peptide.

Any of the above methods of the invention can be practiced, if desired,by selectively determining the presence or absence in the individual ofIgG antibodies immunoreactive with each of the amino acid sequences. Anyof a variety of means can be used to determine the presence or absenceof antibodies immunoreactive with each of the specified amino acidsequences including, yet not limited to, enzyme-linked immunosorbentassays and radioimmunoassays. In one embodiment, the botulinum toxintherapy is BoNT/A therapy.

A variety of assays are useful in a method of the invention fordetermining the presence or absence of antibodies immunoreactive with aBoNT/A peptide including, without limitation, enzyme-linkedimmunosorbent assays and radioimmunoassays. The methods of the inventioncan be useful for predicting or determining immunoresistance to any of avariety of botulinum toxin therapies including, but not limited to,BOTOX® therapy.

The term “immunoresistance,” as used herein in reference to botulinumtoxin therapy, means a reduction in beneficial effect of botulinum toxintherapy in an individual resulting from the presence in the individualof antibodies that bind to botulinum toxin. As used herein, the term“botulinum toxin therapy” means administration to an individual one ormore controlled doses of botulinum toxin to obtain a beneficialtherapeutic or cosmetic effect. The term botulinum toxin therapyencompasses, without limitation, the use of any naturally occurring ormodified or engineered form of a botulinum toxin or a domain or fragmentthereof, in any formulation, combined with any carrier or activeingredient and administered by any route of administration. An exemplarywell-known botulinum toxin therapy is BOTOX® therapy. Appropriatetherapeutic and cosmetic uses of botulinum toxin therapy are known inthe art as discussed above.

A variety of assay formats employing one or more BoNT/A peptides of theinvention can be used to determine the presence or absence of antibodiesimmunoreactive with a BoNT/A and, therefore, to predict or determineimmunoresistance to botulinum toxin therapy according to a method of theinvention. Such assay formats generally involve detecting anantigen-antibody interaction. Non-limiting examples includeradioimmunoassays, enzyme immunoassays, fluorescence immunoassays,luminescent immunoassays and other nonradioisotopic assay formats.Non-competitive assays can be performed, for example, by attaching oneor more selected BoNT/A peptides to a solid support; adding a testspecimen; adding a secondary antibody, which is an antibody selectivefor the test antibody; and detecting the secondary antibody, typicallyby a physical property or enzymatic activity of the secondary antibody.In such an assay, the amount of signal that is detected can beproportional to the amount of antibodies which are immunoreactive withthe one or more BoNT/A peptides and are present in the test specimen.

As a further non-limiting example, a competitive assay can be performedby attaching one or more selected BoNT/A peptides to a solid support;adding simultaneously a test specimen and an enzyme-labeled secondaryantibody; and adding a substrate that produces a detectable compoundwhen acted upon by the enzyme. In this type of assay format, the amountof signal that is detected is inversely proportional to the amount ofBoNT antibody present in the test specimen.

In any assay format selected, a BoNT/A peptide of the inventionoptionally can be attached to a solid support. Such a solid support canbe, without limitation, a tube, plate, column, particle or bead. Thesolid support selected can have a physical property that renders itreadily separable from soluble or unbound material and generally allowsunbound materials, such as unbound antibodies, to be washed away orotherwise removed from support-bound antibodies.

In one embodiment, the presence or absence of antibodies immunoreactivewith a BoNT/A peptide is determined using an enzyme-linked immunosorbentassay (ELISA). In another embodiment, the presence or absence ofantibodies immunoreactive with a BoNT/A peptide is determined using aradioimmunoassay.

It is understood that a method of the invention for predicting ordetermining immunoresistance to botulinum toxin therapy can bedetermined using a test specimen obtained from an individual prior toreceipt of botulinum toxin therapy, after a single botulinum toxintreatment, after multiple botulinum toxin treatments, or after onset ofresistance to botulinum toxin therapy. Useful test specimens include,but are not limited to, serum. It further is understood that a method ofthe invention can be used to predict the likelihood of an individualdeveloping immunoresistance or to confirm that the presence of anti-BoNTantibodies are a cause underlying resistance to botulinum toxin therapy.In particular embodiments, a method of the invention for predicting ordetermining immunoresistance to botulinum toxin therapy in an individualinvolves determining the presence or absence of antibodiesimmunoreactive with two or more BoNT/A peptides, such as, withoutlimitation, five or more BoNT/A peptides, ten or more BoNT/A peptides,or twenty or more BoNT/A peptides. In a further embodiment, a method ofthe invention further includes the step of determining the presence orabsence of antibodies immunoreactive with an Hc peptide.

BoNT/A Tolerogenic Compositions

A BoNT/A peptide of the invention can be combined with another substanceto produce a tolerogizing composition useful for treating an individual.Thus, the present invention further provides a tolerogizing compositioncontaining a tolerogizing agent and a BoNT/A peptide having a length ofat most 60 amino acids that includes the amino acid sequence 445-471 ofSEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 ofSEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 ofSEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 ofSEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 ofSEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 ofSEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or aconservative variant or tolerogenic fragment thereof, with the provisothat the BoNT/A peptide is not SEQ ID NO:2. In one embodiment, theBoNT/A peptide includes the amino acid sequence 515-541 of SEQ ID NO:1,529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 781-807 of SEQ ID NO:1,809-835 of SEQ ID NO:1, or 823-849 of SEQ ID NO:1, or a conservativevariant or tolerogenic fragment thereof, with the proviso that theBoNT/A peptide is not SEQ ID NO:2.

BoNT/A peptides useful in a tolerogizing composition of the inventioncan have, for example, a length of at most 40 amino acids or a length ofat most 25 amino acids. In one embodiment, a tolerogizing composition ofthe invention contains a BoNT/A peptide that includes the amino acidsequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 ofSEQ ID NO:1, or a conservative variant thereof. In another embodiment, atolerogizing composition of the invention contains a BoNT/A peptide thatincludes the amino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQID NO:1, or 837-863 of SEQ ID NO:1, or an immunoreactive fragmentthereof, with the proviso that the BoNT/A peptide is not SEQ ID NO:2. Ina further embodiment, a tolerogizing composition of the inventioncontains a BoNT/A peptide that includes the amino acid sequence 445-471of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1. Instill a further embodiment, a tolerogizing composition of the inventioncontains a BoNT/A peptide that has the amino acid sequence 445-471 ofSEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 ofSEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 ofSEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 ofSEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 ofSEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 ofSEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1. Avariety of tolerogizing agents are useful in the invention including,without limitation, polyethylene glycol (PEG), monomethoxypolyethyleneglycol (mPEG), and polyvinyl alcohol (PVA).

The tolerogizing compositions of the invention are useful, for example,for inducing specific immunological non-reactivity (tolerance) to abotulinum toxin antigen. Tolerance is an active antigen-dependentprocess that occurs in an individual in response to the antigen andresults from a previous exposure to the same antigen. Various moleculesare known in the art to cause, promote or enhance tolerance. See, forexample, U.S. Pat. No. 5,268,454, and citations therein. As used herein,the term “tolerogizing agent” means a molecule, compound or polymer thatcauses, promotes or enhances tolerogenic activity when combined with aBoNT/A peptide of the invention.

A tolerogizing composition of the invention contains one or moretolerogizing agents, which can be present in a variety of forms. Asnon-limiting examples, a tolerogizing agent can be a liquid, solid, oremulsion, depending, for example, on the route of administration andphysical properties of the tolerogizing agent. A tolerogizing agent alsocan be conjugated to a BoNT/A peptide in a tolerogizing composition ofthe invention. Non-limiting examples of tolerogizing agents includepolyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG) andpolyvinyl alcohol (PVA). Such tolerogizing agents can be conjugated to aBoNT/A peptide, for example, as described in Atassi et al., U.S. Pat.No. 6,048,529.

The term “tolerogenic fragment,” as used herein in reference to aportion of SEQ ID NO:1, means a portion of the sequence, or aconservative variant thereof, that has tolerogenic activity as definedby the ability either alone, or in combination with another molecule, toproduce a decreased immunological response. A tolerogenic fragment of aBoNT/A peptide can have from about three amino acids to about 60 aminoacids. A tolerogenic fragment of a BoNT/A peptide can have, for example,at least 5, 8, 10, 12, 15, 18, 20 or 25 amino acids. A tolerogenicfragment of a BoNT/A peptide also can have, for example, at most 8, 10,12, 15, 18, 20, 25, 30 or 35 amino acids. As non-limiting examples, atolerogenic fragment of a BoNT/A peptide can have from five to fiftyamino acids, from eight to fifty amino acids, from ten to fifty aminoacids, from eight to twenty amino acids, from ten to twenty amino acids,from twelve to twenty amino acids or from fifteen to twenty amino acids.

A tolerogenic fragment of a BoNT/A peptide can be identified using anyof a variety of assays, including in vitro assays such as T-cellproliferation or cytokine secretion assays and in vivo assays such asthe induction of tolerance in animal models of botulinum toxicity.T-cell proliferation assays, for example, are well recognized in the artas predictive of tolerogenic activity (see, for example, Miyahara etal., Immunol. 86:110-115 (1995) or Lundin et al, J. Exp. Med.178:187-196 (1993)). A T-cell proliferation assay can be performed, forexample, by culturing T-cells with irradiated antigen-presenting cells,such as normal spleen cells, in microtiter wells for 3 days with varyingconcentrations of the BoNT/A fragment to be assayed; adding³H-thymidine; and measuring incorporation of ³H-thymidine into DNA.

A tolerogenic fragment of a BoNT/A peptide can be identified using aT-cell cytokine secretion assay known in the art. In such an assay, Tcells can be cultured, for example, with irradiated antigen-presentingcells in microtiter wells with varying concentrations of the fragment ofinterest and, after three days, the culture supernatants can be assayedfor IL-2, IL-4 or IFN-γ as described in Czerinsky et al., Immunol. Rev.119:5-22 (1991).

A tolerogenic fragment also can be identified by its ability to inducetolerance in vivo, as indicated by a decreased immunological response,which can be a decreased T-cell response, such as a decreasedproliferative response or cytokine secretion response as describedabove, or a decreased antibody titer to the antigen. A neonatal or adultmouse can be tolerized with a fragment of a BoNT/A peptide, and a T-cellresponse or anti-BoNT/A antibody titer can be assayed after challengingby immunization. As an example, a neonatal mouse can be tolerized within48 hours of birth by intraperitoneal administration of about 100 μg of afragment of a BoNT/A peptide emulsified with incomplete Freund'sadjuvant and subsequently immunized with BoNT/A toxin at about 8 weeksof age (see, for example, Miyahara, supra, 1995). An adult mouse can betolerized intravenously with about 0.33 mg of a fragment of a BoNT/Apeptide, administered daily for three days (total dose 1 mg), andimmunized one week later with BoNT/A. A decreased T-cell response, suchas decreased proliferation or cytokine secretion, which indicatestolerogenic activity, can be measured using T-cells harvested 10 daysafter immunization. In addition, a decreased anti-BoNT/A antibody titer,which also indicates tolerogenic activity, can be assayed using bloodharvested 4-8 weeks after immunization. Methods for assaying a T-cellresponse or anti-BoNT/A antibody titer are described above and are wellknown in the art.

Several well-accepted models of botulinum toxicity can be useful inidentifying a tolerogenic fragment of a BoNT/A peptide. Such modelsinclude, without limitation, rodent, rabbit and monkey models offoodborne botulism, rodent and chicken models of infant botulism androdent models of wound botulism, which are described, for example, inSimpson (Ed.) Botulinum Neurotoxin and Tetanus Toxin Academic Press,Inc., San Diego, Calif. (1989). The skilled person understands thatthese and a variety of other well known in vitro and in vivo assays canbe useful for identifying a tolerogenic fragment of a BoNT/A peptide.

Preventing or Reducing Immunoresistance to BoNT Therapy

The BoNT/A peptides of the invention also can be useful for preventingor reducing development of a BoNT-specific immune response in anindividual, which in turn can prevent or reduce immunoresistance tobotulinum toxin therapy. Thus, the present invention provides a methodof preventing or reducing immunoresistance to botulinum toxin therapy inan individual by administering to the individual a tolerogizing agentand a BoNT/A peptide having a length of at most 60 amino acids andcontaining the amino acid sequence 445-471 of SEQ ID NO:1, 487-513 ofSEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 ofSEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 ofSEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 ofSEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 ofSEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 ofSEQ ID NO:1, or 837-863 of SEQ ID NO:1, or a conservative variant orimmunoreactive fragment thereof, thereby preventing or reducingimmunoresistance to botulinum toxin therapy, with the proviso that theBoNT/A peptide is not SEQ ID NO:2.

BoNT/A peptides useful for preventing or reducing immunoresistance tobotulinum toxin therapy according to a method of the invention can have,for example, a length of at most 40 amino acids or a length of at most25 amino acids. In one embodiment, a BoNT/A peptide useful forpreventing or reducing immunoresistance includes the amino acid sequence445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1,529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1,585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1,669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1,739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1,809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ IDNO:1, or a conservative variant thereof. In another embodiment, a BoNT/Apeptide useful for preventing or reducing immunoresistance includes theamino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1,515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1,711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1,or 837-863 of SEQ ID NO:1, or an immunoreactive fragment thereof, withthe proviso that the BoNT/A peptide is not SEQ ID NO:2. In a furtherembodiment, a BoNT/A peptide useful for preventing or reducingimmunoresistance includes the amino acid sequence 445-471 of SEQ IDNO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ IDNO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ IDNO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ IDNO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ IDNO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ IDNO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1. In still afurther embodiment, a BoNT/A peptide useful for preventing or reducingimmunoresistance has the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1. In a furtherembodiment, a method of the invention is practiced by administering thetolerogizing agent and BoNT/A peptide prior to the individual receivingbotulinum toxin therapy. Such an individual can be, for example, anindividual at increased risk for developing immunoresistance tobotulinum toxin therapy.

The present invention further provides a method of preventing orreducing immunoresistance to botulinum toxin therapy in an individual byadministering to the individual a tolerogizing agent and two or more ofthe following amino acid sequences: 785-803 of SEQ ID NO: 1 [N25];981-999 of SEQ ID NO: 1 [C10]; 1051-1069 of SEQ ID NO: 1 [C15];1121-1139 of SEQ ID NO: 1 [C20]; and 1275-1296 of SEQ ID NO: 1 [C31], ora conservative variant or an immunoreactive fragment of any of thesesequences, thereby preventing or reducing immunoresistance to botulinumtoxin therapy. In one embodiment, one of the amino acid sequencesincludes residues 785-803 of SEQ ID NO: 1 [N25] or a conservativevariant or immunoreactive fragment of this sequence. In anotherembodiment, a method of the invention is practiced by administering tothe individual a tolerogizing agent and the following two amino acidsequences: 785-803 of SEQ ID NO: 1 [N25]; and 981-999 of SEQ ID NO: 1[C10], or a conservative variant or immunoreactive fragment of any ofthese sequences. In a further embodiment, a method of the invention ispracticed by administering to the individual a tolerogizing agent andthe following three amino acid sequences: 785-803 of SEQ ID NO: 1 [N25];981-999 of SEQ ID NO: 1 [C10]; and 1051-1069 of SEQ ID NO: 1 [C15], or aconservative variant or an immunoreactive fragment of any of thesesequences. Again, the two or more amino acid sequences can be providedseparately or as part of a compound molecule such as a chimericsynthetic peptide. The methods of the invention can be useful forpreventing or reducing immunoresistance to any of a variety of botulinumtoxin therapies including, but not limited to, BoNT/A therapy.

A tolerogizing agent and BoNT/A peptide can be administered to anindividual prior to administering botulinum toxin therapy to prevent thedevelopment of immunoresistance, during a course of botulinum toxintherapy, or after onset of immunoresistance, for example, when symptomsof resistance are first apparent. In addition, a tolerogizing agent andBoNT/A peptide can be administered to an individual who is at increasedrisk for immunoresistance to botulinum toxin therapy. Those skilled inthe art will be able to determine an appropriate candidate for receivinga tolerogizing composition of the invention based on, for example, theparticular condition to be treated and the presence or likelihood ofsymptoms of immunoresistance.

A tolerogizing agent and BoNT/A peptide can be formulated in a varietyof pharmaceutically acceptable media, described below. An effective doseof a BoNT/A peptide of the invention for inducing tolerance in anindividual will depend upon the particular BoNT/A peptide selected, thetolerogizing agent used, the route administration, and the particularcharacteristics of the individual, such as age, weight, general healthand the like. An effective dose can be determined in an animal model,such as one of those described hereinabove, prior to administration tohumans.

Tolerogizing agents and BoNT/A peptides useful in the invention can beadministered by a variety of routes to stimulate an immune response. Asa non-limiting example, oral tolerance is well-recognized in the art(see, for example, Weiner, Hospital Practice, pp. 53-58 (Sep. 15,1995)).

BoNT/A Peptide Vaccines

The present invention further provides vaccine compositions containingan adjuvant and a BoNT/A peptide of the invention. In particular, theinvention provides a vaccine composition that contains an adjuvant and aBoNT/A peptide having a length of at most 60 amino acids and includingthe amino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1,515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1,711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1,or 837-863 of SEQ ID NO:1, or a conservative variant or immunoreactivefragment thereof, with the proviso that the BoNT/A peptide is not SEQ IDNO:2. In one embodiment, the BoNT/A peptide includes the amino acidsequence 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQID NO:1, 585-611 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 739-765 of SEQID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, or 823-849 ofSEQ ID NO:1, or a conservative variant or immunoreactive fragmentthereof, with the proviso that the BoNT/A peptide is not SEQ ID NO:2.

In a vaccine composition of the invention, the BoNT/A peptide can have,for example, a length of at most 40 amino acids or a length of at most25 amino acids. In one embodiment, the vaccine composition contains aBoNT/A peptide that includes the amino acid sequence 445-471 of SEQ IDNO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ IDNO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ IDNO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ IDNO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ IDNO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ IDNO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or aconservative variant thereof. In another embodiment, the vaccinecomposition contains a BoNT/A peptide that includes the amino acidsequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 ofSEQ ID NO:1, or an immunoreactive fragment thereof, with the provisothat the BoNT/A peptide is not SEQ ID NO:2. In a further embodiment, thevaccine composition contains a BoNT/A peptide that includes the aminoacid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 ofSEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 ofSEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 ofSEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 ofSEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 ofSEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863of SEQ ID NO:1. In still a further embodiment, the vaccine compositioncontains a BoNT/A peptide that has the amino acid sequence 445-471 ofSEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 ofSEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 ofSEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 ofSEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 ofSEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 ofSEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1.

The vaccines of the invention can be useful, for example, for inducingspecific immunity against one or more botulinum toxins such as BoNT/A.Such specific immunity can protect an individual from intoxicationproduced by exposure to botulinum toxin. As used herein, the term“vaccine” means a composition which, when administered to an individual,stimulates an immune response against an antigen. A vaccine can beuseful, for example, for preventing or ameliorating intoxicationproduced by unwanted exposure to botulinum toxin. Vaccination usingpeptides has been shown to effectively block the effect of proteintoxins. See, for example, Dolimbek et al., J. Prot. Chem. 13:490 (1994);Atassi et al., Mol. Immunol. 13:927 (1995); and Dolimbek et al., Mol.Immunol. 33:681 (1996).

A vaccine of the invention contains one or more BoNT/A peptide antigens.The BoNT/A peptides included in a vaccine of the invention can beselected, for example, depending on immunological factors, such aspotency of the peptide in inducing an immune response, and technicalfactors, such as chemical synthesis yields. A vaccine of the inventionalso contain one or more adjuvants. The term “adjuvant” as used herein,means a substance that acts generally to accelerate, prolong, or enhancethe quality of a specific immune response to a vaccine antigen. Anadjuvant can, for example, serve to reduce the number of immunizationsor the amount of antigen required for protective immunization. Asnon-limiting examples, an adjuvant useful in the invention can be analuminum salt based adjuvant or an immunomodulatory compound such asGM-CSF.

A vaccine of the invention can include a BoNT/A peptide which is, forexample, conjugated to, or expressed as a fusion protein with anothermolecule. The molecule selected for fusion to a BoNT/A peptide willdepend on the particular design of the vaccine. Non-limiting examples ofBoNT/A fusion proteins useful in the invention include fusions withmolecules that increase immune response against the BoNT/A peptide, suchas cholera enterotoxin A2 and other peptides against which an immuneresponse is desired, such as another BoNT peptide. In one embodiment, avaccine of the invention contains a BoNT/A peptide fused to a peptide orprotein adjuvant.

Method for Vaccinating an Individual Against BoNT

A vaccine of the invention can stimulate an immune response againstbotulinum toxin in an individual, resulting in the production ofantibodies that bind to and neutralize botulinum toxin. Such an immuneresponse increases the ability of an individual's immune system todestroy botulinum toxin and thereby prevent harmful effects of botulinumtoxin exposure.

Thus, the present invention provides a method of vaccinating anindividual against botulinum toxin by administering to the individual avaccine containing an adjuvant and a BoNT/A peptide which has a lengthof at most 60 amino acids and contains the amino acid sequence 445-471of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or aconservative variant or immunoreactive fragment thereof, therebyproducing an immune response to botulinum toxin in the individual, withthe proviso that the BoNT/A peptide is not SEQ ID NO:2. In oneembodiment, a method of the invention is practiced using a BoNT/Apeptide that contains the amino acid sequence 515-541 of SEQ ID NO:1,529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 781-807 of SEQ ID NO:1,809-835 of SEQ ID NO:1, or 823-849 of SEQ ID NO:1, or a conservativevariant or immunoreactive fragment thereof, with the proviso that theBoNT/A peptide is not SEQ ID NO:2.

BoNT/A peptides useful for vaccinating an individual against botulinumtoxin according to a method of the invention can have, for example, alength of at most 40 amino acids or a length of at most 25 amino acids.In one embodiment, the BoNT/A peptide includes the amino acid sequence445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1,529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1,585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1,669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1,739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1,809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ IDNO:1, or a conservative variant thereof. In another embodiment, theBoNT/A peptide includes the amino acid sequence 445-471 of SEQ ID NO:1,487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1,543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1,683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1,823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1, or an immunoreactivefragment thereof, with the proviso that the BoNT/A peptide is not SEQ IDNO:2. In a further embodiment, the BoNT/A peptide includes the aminoacid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 ofSEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 ofSEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 ofSEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 ofSEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 ofSEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863of SEQ ID NO:1. In still a further embodiment, the BoNT/A peptide hasthe amino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1,515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1,711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1,or 837-863 of SEQ ID NO:1.

Further provided herein are methods of vaccinating an individual againstbotulinum toxin by administering to the individual a vaccine containingan adjuvant and two or more of the following amino acid sequences:785-803 of SEQ ID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10]; 1051-1069of SEQ ID NO: 1 [C15]; 1121-1139 of SEQ ID NO: 1 [C20]; and 1275-1296 ofSEQ ID NO: 1 [C31], or a conservative variant or an immunoreactivefragment of one of these sequences, thereby producing an immune responseto the botulinum toxin in the individual. In one embodiment, one of theamino acid sequence includes residues 785-803 of SEQ ID NO: 1 [N25] or aconservative variant or immunoreactive fragment of this sequence. Inanother embodiment, the vaccine includes an adjuvant and the followingtwo amino acid sequences: 785-803 of SEQ ID NO: 1 [N25]; and 981-999 ofSEQ ID NO: 1 [C10], or a conservative variant or immunoreactive fragmentof any of these sequences. In a further embodiment, the vaccine includesan adjuvant and the following three amino acid sequences: 785-803 of SEQID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10]; and 1051-1069 of SEQ IDNO: 1 [C15], or a conservative variant or an immunoreactive fragment ofany of these sequences. As for the methods disclosed above, the two ormore amino acid sequences can be provided separately or as part of acompound molecule such as a chimeric synthetic peptide.

Thus, the present invention provides a method of removing botulinumtoxin blocking antibodies from a patient by removing blood from apatient; contacting the blood, or an antibody-containing componentthereof, with two or more of the following amino acid sequences: 785-803of SEQ ID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10]; 1051-1069 of SEQID NO: 1 [C15]; 1121-1139 of SEQ ID NO: 1 [C20]; and 1275-1296 of SEQ IDNO: 1 [C31], or a conservative variant or an immunoreactive fragmentthereof, under conditions suitable for forming a complex of each of theamino acid sequences and anti-botulinum toxin antibody; and removing thecomplex from the blood or antibody-containing component thereof. In oneembodiment, patient blood, or an antibody-containing component thereof,is contacted with the following two amino acid sequences: 785-803 of SEQID NO: 1 [N25]; and 981-999 of SEQ ID NO: 1 [C10], or a conservativevariant or an immunoreactive fragment of any of these sequences. Inanother embodiment, patient blood, or an antibody-containing componentthereof, is contacted with the following three amino acid sequences:785-803 of SEQ ID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10]; and1051-1069 of SEQ ID NO: 1 [C15], or a conservative variant or animmunoreactive fragment of one of these sequences. It is understood thatany of the above methods of removing botulinum toxin blocking antibodiesfrom a patient can be practiced by selectively removing IgGanti-botulinum toxin antibodies. It is further understood that the twoor more amino acid sequences can be provided separately or as part of acompound molecule such as a chimeric synthetic peptide.

A vaccine useful in a method of the invention can be administered by anyof a variety of routes, as described below in relation to pharmaceuticalcompositions. Those skilled in the art can readily determine for aparticular BoNT/A vaccine, the appropriate antigen payload; route ofimmunization; volume of dose; and vaccination regimen useful in aparticular animal, for example, humans.

One skilled in the art can determine if a BoNT/A vaccine induces animmune response, as methods for detecting immune responses are wellknown in the art. Non-limiting examples involve measuring the titer ofBoNT/A-selective antibodies in an animal primed with the vaccine andboosted with the antigen, or determining the presence of antibodies inthe blood of an immunized animal that are cross-reactive with theantigen by ELISA, Western blotting or other well-known methods.Cell-mediated immune responses can be determined, for example, bymeasuring cytotoxic T cell response to antigen using a variety ofmethods described hereinabove or well known in the art.

BoNT/A Antibodies

A BoNT/A peptide of the invention can be used in a process for preparingan anti-BoNT antibody. Thus, the present invention provides a method ofpreparing an anti-BoNT/A antibody by administering to an animal a BoNT/Apeptide having a length of at most 60 amino acids and containing theamino acid sequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1,515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1,711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1,or 837-863 of SEQ ID NO:1, or a conservative variant or immunoreactivefragment thereof; collecting from the animal a sample containing anantibody or antibody-producing cell; and processing the sample toisolate the anti-BoNT/A antibody, with the proviso that the BoNT/Apeptide is not SEQ ID NO:2. In one embodiment, a method of the inventionis practiced with a BoNT/A peptide containing the amino acid sequence515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1,585-611 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 739-765 of SEQ ID NO:1,781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, or 823-849 of SEQ IDNO:1, or a conservative variant or immunoreactive fragment thereof, withthe proviso that the BoNT/A peptide is not SEQ ID NO:2.

BoNT/A peptides useful for preparing an anti-BoNT/A antibody can have,for example, a length of at most 40 amino acids or a length of at most25 amino acids. In one embodiment, a method of the invention ispracticed by administering a BoNT/A peptide that includes the amino acidsequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 ofSEQ ID NO:1, or a conservative variant thereof. In another embodiment, amethod of the invention is practiced by administering a BoNT/A peptidethat includes the amino acid sequence 445-471 of SEQ ID NO:1, 487-513 ofSEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ ID NO:1, 543-569 ofSEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ ID NO:1, 599-625 ofSEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ ID NO:1, 683-709 ofSEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 767-793 ofSEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ ID NO:1, 823-849 ofSEQ ID NO:1, or 837-863 of SEQ ID NO:1, or an immunoreactive fragmentthereof, with the proviso that the BoNT/A peptide is not SEQ ID NO:2. Ina further embodiment, a method of the invention is practiced byadministering a BoNT/A peptide that includes the amino acid sequence445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1,529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1,585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1,669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1,739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1,809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ IDNO:1. In yet a further embodiment, a method of the invention ispracticed by administering a BoNT/A peptide that has the amino acidsequence 445-471 of SEQ ID NO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQID NO:1, 529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQID NO:1, 585-611 of SEQ ID NO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQID NO:1, 669-695 of SEQ ID NO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQID NO:1, 739-765 of SEQ ID NO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQID NO:1, 809-835 of SEQ ID NO:1, 823-849 of SEQ ID NO:1, or 837-863 ofSEQ ID NO:1.

Antibodies to be prepared according to a method of the invention includepolyclonal and monoclonal antibodies. A BoNT/A antibody preparedaccording to a method of the invention, or a monoclonal anti-BoNT/Aantibody of the invention as described further below, can be used in avariety of applications. Such applications include, for example,detection of botulinum toxin in a sample, such as a substance suspectedto be contaminated with BoNT/A.

As used herein, the term “antibody” includes polyclonal and monoclonalantibodies, as well as polypeptide fragments of antibodies thatselectively bind to a BoNT polypeptide. Such selective binding refers tothe discriminatory binding of the antibody to the indicated targetpeptide or polypeptide such that the antibody does not substantiallycross react with unrelated peptides or polypeptides. Specific reactivitycan include binding properties such as binding specificity, bindingaffinity and binding avidity. For example, an antibody can bind a targetpeptide or polypeptide with a binding affinity (Kd) of about 10⁻⁴ M ormore, 10⁻⁶ M or more, 10⁻⁷ M or more, 10⁻⁸ M or more, 10⁻⁹ M or more, or10⁻¹⁰ M or more. Several methods for detecting or measuring antibodybinding are known in the art and disclosed herein. Monoclonal antibodiesrefer to a population of antibody molecules that contain only onespecies of antibody capable of binding a particular antigen. Methods ofproducing a monoclonal antibody are well known (see, for example, Harlowand Lane, supra, 1988). As a non-limiting example, a hybridoma thatproduces a BoNT/A monoclonal antibody can be identified by screeninghybridoma supernatants for the presence of antibodies that bind to aBoNT/A peptide of the invention (Harlow, supra, 1988). For example,hybridoma supernatants can be screened using BoNT/A-positive sera in aradioimmunoassay or enzyme-linked immunosorbent assay. Polyclonalantibodies refer to a population of antibody molecules that contain twoor more species of antibody capable of binding to a particular antigen.Methods of producing a polyclonal antibody are well known (see, forexample, Harlow and Lane, supra, 1988). As a non-limiting example, serumfrom an animal immunized with a BoNT/A peptide of the invention can bescreened in a radioimmunoassay or enzyme-linked immunosorbent assay toidentify a polyclonal BoNT/A antibody.

A variety of well known methods can be used for collecting from ananimal a sample containing an antibody or antibody-producing cell. Suchmethods are described, for example, in Harlow et al., supra, 1998.Similarly, a variety of well known methods can be used for processing asample to isolate an anti-BoNT/A antibody. A procedure for collectingand processing a sample can be selected based on the type of antibody tobe isolated. As an example, when isolating polyclonal antibodies, anappropriate sample can be a blood sample containing antibodies, whereaswhen isolating monoclonal antibodies, an appropriate sample can be anantibody-producing cell such as a spleen cell. Exemplary well knownprocedures for isolating both monoclonal and polyclonal antibodies areknown in the art as described above.

Monoclonal Anti-BoNT/A Antibodies

In another embodiment, the present invention provides an anti-BoNT/Amonoclonal antibody having specificity for an epitope contained withinone of the following amino acid sequences: amino acids 445-471 of SEQ IDNO:1, 487-513 of SEQ ID NO:1, 515-541 of SEQ ID NO:1, 529-555 of SEQ IDNO:1, 543-569 of SEQ ID NO:1, 557-583 of SEQ ID NO:1, 585-611 of SEQ IDNO:1, 599-625 of SEQ ID NO:1, 655-681 of SEQ ID NO:1, 669-695 of SEQ IDNO:1, 683-709 of SEQ ID NO:1, 711-737 of SEQ ID NO:1, 739-765 of SEQ IDNO:1, 767-793 of SEQ ID NO:1, 781-807 of SEQ ID NO:1, 809-835 of SEQ IDNO:1, 823-849 of SEQ ID NO:1, or 837-863 of SEQ ID NO:1. In a furtherembodiment, the present invention provides an anti-BoNT/A monoclonalantibody having specificity for an epitope contained within one of thefollowing amino acid sequences: amino acids 515-541 of SEQ ID NO:1,529-555 of SEQ ID NO:1, 543-569 of SEQ ID NO:1, 585-611 of SEQ ID NO:1,655-681 of SEQ ID NO:1, 739-765 of SEQ ID NO:1, 781-807 of SEQ ID NO:1,809-835 of SEQ ID NO:1, or 823-849 of SEQ ID NO:1. In yet a furtherembodiment, the present invention provides an anti-BoNT/A monoclonalantibody having specificity for an epitope contained within amino acids785-803 of SEQ ID NO: 1, which corresponds to the epitope defined withinthe N25 peptide. In still further embodiments, any of the monoclonalantibodies disclosed above are of the IgG subtype. As used herein, theterm “monoclonal antibody” means a homogeneous population of antibodyspecies. By definition, a monoclonal antibody binds to a single epitope.

Treatment of Botulinum Toxicity

A BoNT/A antibody prepared according to a method of the invention canbind to a botulinum toxin and neutralize its effects. Thus, the presentinvention provides a method of treating botulinum toxicity in anindividual by administering to the individual a pharmaceuticalcomposition containing an anti-BoNT/A antibody produced according to amethod of the invention or an anti-BoNT/A monoclonal antibody of theinvention. Botulinum toxicity refers to intoxication resulting fromexposure to botulinum toxin. Botulism clinical syndromes include foodborne botulism, which can result from ingestion of preformed botulinumtoxin in contaminated foods; wound botulism, which can result from theproduction of botulinum toxin in vivo after growth of C. botulinum in aninfected wound; GI colonization syndromes, which can result from theproduction of botulinum toxin in vivo due after growth of C. botulinumin the intestinal tract of a colonized individual; iatrogenic botulism,which can result from injection of botulinum toxin into a tissue of anindividual; and inhalation botulism, which can occur accidentally inhumans, for example, in a veterinary setting when working with infectedanimals, and as a result of biological warfare. The signs and symptomsof botulinum intoxication are well known to those skilled in the art.

Experiments performed in vivo and in vitro have indicated thatantibodies can enter cholinergic nerves and neutralize internalized BoNT(Simpson, J. Physiol. Paris 84:143 (1990)). As such, anti-BoNTantibodies can act, for example, extracellularly by interfering with thebinding of BoNT to the cell surface and intracellularly by interferingwith BoNT enzymatic activity. The ability of an anti-BoNT/A antibodyprepared according to a method of the invention to neutralize theeffects of botulinum toxicity on an individual, and, thus, “protectagainst” botulinum toxicity, can be determined in an animal model usinga variety of methods well known to those skilled in the art. Exemplaryanimal models of botulism include rodent, rabbit and monkey models offoodborne botulism, rodent and chicken models of infant botulism androdent models of wound botulism, all of which are described, forexample, in Simpson, supra, 1989.

Plasmapheresis

The BoNT/A peptides disclosed herein also can be useful for therapeuticimmunoadsorption for extracorporeal removal of anti-botulinum toxinantibodies. Such therapeutic immunoadsorption is well known in the art.In general, blood can be removed from a patient to be treated or havingbeen treated with a botulinum toxin therapeutic such as BOTOX®; andanti-botulinum toxin antibodies subsequently removed from the blood,serum or plasma using affinity chromatography with one or more BoNT/Apeptides of the invention are attached to a biocompatible support. Inone embodiment, an N25 BoNT/A peptide is used for therapeuticimmunoadsorption such that anti-N25 antibodies are removed from patientblood, serum or plasma. In another embodiment, one or a combination ofN25, C10, C15, C20 or C31 BoNT/A peptides are used for therapeuticimmunoadsorption such that antibodies to epitopes in the peptides usedfor the immunoadsorption are removed from patient blood, serum orplasma.

Thus, the present invention provides a method of removing botulinumtoxin blocking antibodies from a patient by removing blood from apatient; contacting the blood, or an antibody-containing componentthereof, with two or more of the following amino acid sequences: 785-803of SEQ ID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10]; 1051-1069 of SEQID NO: 1 [C15]; 1121-1139 of SEQ ID NO: 1 [C20]; and 1275-1296 of SEQ IDNO: 1 [C31], or a conservative variant or an immunoreactive fragmentthereof, under conditions suitable for forming a complex of each of theamino acid sequences and anti-botulinum toxin antibody; and removing thecomplex from the blood or antibody-containing component thereof. In oneembodiment, patient blood, or an antibody-containing component thereof,is contacted with the following two amino acid sequences: 785-803 of SEQID NO: 1 [N25]; and 981-999 of SEQ ID NO: 1 [C10], or a conservativevariant or an immunoreactive fragment of any of these sequences. Inanother embodiment, patient blood, or an antibody-containing componentthereof, is contacted with the following three amino acid sequences:785-803 of SEQ ID NO: 1 [N25]; 981-999 of SEQ ID NO: 1 [C10]; and1051-1069 of SEQ ID NO: 1 [C15], or a conservative variant or animmunoreactive fragment of one of these sequences. It is understood thatany of the above methods of removing botulinum toxin blocking antibodiesfrom a patient can be practiced by selectively removing IgGanti-botulinum toxin antibodies. It is further understood that the twoor more amino acid sequences can be provided separately or as part of acompound molecule such as a chimeric synthetic peptide.

Biocompatible solid supports having combinations of two or more BoNT/Apeptides can be useful in plasma or other pheresis, or pheresis can beperformed using a series of affinity columns or other solid supportseach having a different BoNT/A peptide. It is understood that the blood,serum or plasma are contacted with the one or more BoNT/A peptidesattached to a biocompatible solid support under conditions that promotebinding between the one or more BoNT/A peptides and anti-botulinum toxinantibodies in the patient fluid. As an example, extracorporealhemoperfusion can be performed as described in U.S. Pat. No. 5,149,425.Such conditions can include, without limitation, contact temperatures inthe range of 35° C. and 40° C., and contact times of about one to sixhours. It is understood that the unbound portion of the blood, plasma,or serum, which is significantly antibody-depleted, is reintegrated withcellular components of whole blood as necessary and reintroduced intothe patient on a continuous basis or following collection. One skilledin the art further understands that, if desired, the antibody-depletedblood, plasma or serum can be assayed prior to reintroduction in thepatient, for example, using one of the BoNT/A peptide binding assays orprotection assays disclosed herein.

Several techniques can be useful for removing anti-botulinum toxinblocking antibodies complexed with a BoNT/A peptide. As an example, asolid phase system can utilize a solid phase matrix which is a solidphrase support to which the one or more BoNT/A peptides are bound. Theblood, plasma or serum containing the blocking antibodies is passed overthe solid support, exiting the solid support and leaving behind theblocking antibody/peptide complexes. A variety of biocompatible solidsupports can be useful in the methods of the invention. Such supportsare chemically inert with respect to human antibody-containing fluids,have sufficient binding capacity, and generally are in the form of acontinuous large surface such as a sheet or column, or in the form ofparticles or vesicles. Exemplary solid supports useful in the invention,including those useful for affinity chromatography, encompass, withoutlimitation, silica; synthetic silicates such as porous glass, forexample, glass fiber filters; biogenic silicates such as diatomaceousearth; silicate-containing materials such as kaolinite and borosilicate;and synthetic polymers such as polystyrene, polypropylene andpolysaccharides (see, for example, U.S. Pat. Nos. 6,607,723 and5,149,425. Biocompatible solid supports useful in the invention furtherinclude, yet are not limited to, agarose, which is a neutral linearpolysaccharide generally composed of D-galactose and altered3,6-anhydrogalactose residues, for example, Sepharose (Pharmacia);activated gels, cellulose, nitrocellulose, polyvinylchloride, anddiazotized paper. The skilled person understands that these and avariety of other well known biocompatible solid supports can be usefulin the methods of the invention.

The one or more BoNT/A peptides can be covalently or noncovalently boundto the solid support using well known methods. Supports which can benon-covalently bound by incubation with the immunosorbent include,without limitation, nitrocellulose, borosilicate, filters,polyvinylchloride, polystyrene and diazotized paper. Activated solidsupports such as activated matrices also are well known in the art andcommercially available and useful in the invention. Such activated solidsupports encompass, without limitation, epoxy-activated agarose;CNBr-activated agarose; 6-aminohexanoic acid and1,6-diaminohexane-agarose, thiopropyl agarose;carbonyldiimidazole-activated agarose; and aminoethyl andhydrazide-activated polyacrylamide (see, for example, U.S. Pat. Nos.6,406,861 and 4,762,787).

In one embodiment, the methods of the invention for selectively removingblocking anti-botulinum toxin antibodies are performed using an affinitycolumn. An affinity column is a cylindrical container with filters onboth ends which contains a solid support to which the one or more BoNT/Apeptides are bound. One skilled in the art understands that plasma orserum generally is passed through a column since whole blood containscells and particulate matter such as platelets which can impede columnflow. In another embodiment, a sheet such as a nitrocellulose sheet ispre-bound with one or more BoNT/A peptides, and blood, plasma or serumis incubated with the immunosorbent-linked nitrocellulose. In a furtherembodiment, one or more BoNT/A peptides are bound to large polystyrenepetri dishes. Blood, plasma or serum from an individual is incubatedwith the BoNT/A peptide-linked polystyrene and is decanted, leavingbehind the blocking antibodies complexed to the one or more BoNT/Apeptides.

It is further understood that pre-clearance of antibodies, or a class ofantibody such as the IgG class, can be performed prior to selectiveremoval of anti-botulinum toxin antibodies. From the pre-clearedantibody pool, BoNT/A peptide-reactive antibodies can be selected, andthe remaining antibodies reconstituted into the blood to be reperfusedinto the individual, thus reducing the volume to be passed over theblocking antibody selective support and also reducing non-specificbinding. As a non-limiting example, non-specific Protein G Sepharosecolumns such as PROSORBA® (IMRE; Munich, Germany) or Ig-THERASORB®(Plasmaselect; Teterow, Germany) can be used to remove a significantportion of IgG antibody. A variety of additional techniques suitable forgeneral pre-clearance of antibodies are well known in the art andinclude, yet are not limited to, ammonium sulfate precipitation with ionexchange chromatography; caprylic acid; DEAE-matrices (ion-exchangechromatography); hydroxyapatite chromatography, and gel filtration(Sepharose). See, for example, Harlow and Lane, supra, 1998.

In still a further embodiment, one or more BoNT/A peptides are bound tolipid vesicles, and the lipid vesicle-immunosorbent is mixed with apatient's plasma or serum to allow binding to the blocking antibodies.The plasma or serum is subsequently filtered to remove the lipidvesicle-immunosorbent-antibody complex. See, for example, U.S. Pat. No.4,643,718.

One skilled in the art further understands that one or more BoNT/Apeptides of the invention can be used for liquid phase separation ofblocking antibodies from patient blood, plasma or serum. Liquid phaseseparation can be performed, for example, by conjugating one or moreBoNT/A peptides to a hapten such as, without limitation, dinitrophenolor fluorescein. After mixing the hapten/BoNT/A peptide conjugate with apatient's blood, plasma or serum, the conjugate forms complexes withanti-botulinum toxin blocking antibodies. As a non-limiting example,such antibody complexes can be precipitated using polyethylene glycol(PEG), and the precipitated complexes separated from the blood, plasmaor serum using centrifugation (see, for example, U.S. Pat. No.4,551,435). One skilled in the art appreciates that these and othersolid-phase and liquid-phase systems can be used to separate BoNT/Apeptide/blocking antibody complexes from patient blood, plasma or serum.

Blocking Neutralizing Antibodies In Situ

As disclosed herein in Example IX and discussed above, one or more ofthe synthetic peptides N25, C10, N15, N20 or N31 binds protectiveantibodies in the large majority of protective patient sera in a sampleof 28 cervical dystonia patients treated with BOTOX® and havingMPA-protective sera. Based on this finding, one or more of the BoNT/Apeptides N25, C10, N15, N20 or N31, or a conservative variant orimmunoreactive fragment thereof, can be useful for decreasing patientnon-responsiveness when administered in excess together with atherapeutic botulinum toxin preparation.

Increased IgG Levels

The present invention additionally provides a method of predicting ordetermining immunoresistance to botulinum toxin therapy in an individualby determining the level of IgG antibodies immunoreactive with thebotulinum toxin in the individual; and comparing the level of IgGantibodies to a control level of IgG antibodies, where an increase inthe level of IgG antibodies in the individual as compared to the controllevel indicates immunoresistance to the botulinum toxin therapy. Such anincrease can be, for example, at least a 5-fold increase or at least a10-fold increase. In one embodiment, the control level of IgG antibodiesis determined in an individual who has not been treated with botulinumtoxin therapy. In another embodiment, the control level of IgGantibodies is determined in an individual who is responsive to thebotulinum toxin therapy. The methods of the invention can be used topredict or determine immunoresistance to any of several botulinum toxintherapies including, without limitation, BoNT/A therapy.

Techniques for determining a level of IgG antibodies immunoreactive witha botulinum toxin such as BoNT/A are well known in the art and aredescribed herein. For example, Example VIII describes a solid-phaseradioimmunoassay for IgG anti-BoNT/A antibodies using an anti-mouse IgGsecondary antibody. A variety of additional anti-IgG antibodies,including anti-human IgG antibodies, are well known in the art and arecommercially available, including, but not limited to, rabbit anti-humanIgG from Bethyl Laboratories, Inc. (Montgomery, Tex.) and goatanti-human IgG from Zymed Laboratories, Inc (San Francisco, Calif.).Thus, the methods of the invention can be practiced using any of theimmunoassays described hereinabove or well known in the art which arespecific for detection of IgG antibodies, for example, through use of ananti-IgG secondary antibody.

Pharmaceutical Compositions

A vaccine composition containing a BoNT/A peptide, tolerogeniccomposition containing a BoNT/A peptide, or a BoNT/A antibody of theinvention can be prepared as a pharmaceutical composition for use in atherapeutic method of the invention. A pharmaceutical composition caninclude an excipient well known in the art for preparing pharmaceuticalcompositions, including compositions suitable for intranasal and oraladministration. A pharmaceutical composition includes a pharmaceuticallyacceptable carrier, which is any carrier that has substantially no longterm or permanent detrimental effect when administered. Examples ofpharmaceutically acceptable carriers include, without limitation, water,such as distilled or deionized water; saline; and other aqueous media.It is understood that the active ingredients can be soluble or can bedelivered as a suspension in a suitable carrier.

A preservative or tonicity adjustor can be included, if desired, in apharmaceutical composition useful in the invention. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, and phenylmercuric nitrate. Tonicityadjustors useful in the invention include salts such as sodium chloride,potassium chloride, mannitol or glycerin and other pharmaceuticallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH can be used to prepare apharmaceutical composition useful in the invention, provided that theresulting preparation is pharmaceutically acceptable. Such buffersinclude, without limitation, acetate buffers, citrate buffers, phosphatebuffers and borate buffers. It is understood that acids or bases can beused to adjust the pH of the composition as needed. Pharmaceuticallyacceptable antioxidants useful in the invention include, yet are notlimited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene.

A variety of routes of administration can be useful in the inventiondepending, in part, on the size and characteristics of the BoNT/Apeptide, tolerogenic composition, vaccine composition, or antibody to beadministered and the history, risk factors and symptoms of the subjectto be treated. Routes of administration suitable for the methods of theinvention include both systemic and local administration.

Exemplary routes of administration useful in the methods of theinvention encompass, without limitation, oral delivery; intravenousinjection; intramuscular injection; subcutaneous injection;intraperitoneal injection; transdermal diffusion and electrophoresis;topical eye drops and ointments; periocular and intraocular injectionincluding subconjunctival injection; extended release delivery devicesincluding locally implanted extended release devices including abioerodible or reservoir-based implants. It is understood that animplant useful in the invention generally releases the implantedpharmaceutical composition over an extended period of time.

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are also includedwithin the definition of the invention provided herein. Accordingly, thefollowing examples are intended to illustrate but not limit the presentinvention.

EXAMPLE I Mapping of Human Anti-Pentavalent Botulinum Toxoid AntibodiesUsing BoNT/A Synthetic Peptides

This example shows antigenic mapping of botulinum toxin A with humananti-BoNT antisera using 29 BoNT/A synthetic peptides that encompass theH_(N) domain of BoNT/A.

Human antisera against BoNT/A were prepared by immunizing humanvolunteers with a toxoid preparation made from BoNTs A, B, C, D and E asdescribed in Atassi et al. supra, 1996. The binding assays describedbelow were performed using IgG fractions of these antisera. For use as acontrol, an IgG fraction was prepared using pre-immune human serum.

For use in antigenic mapping, BoNT/A peptides were synthesized, purifiedand subjected to amino acid analysis by the procedure previouslyreported (Atassi et al., Proc. Natl. Acad. Sci. USA 88:3613 (1991)).Each peptide was found to have an amino acid composition consistent withthat expected from its covalent structure shown in FIG. 1. BoNTs A and Bwere purchased from Metabiologics (Madison, Wis.).

BoNT/A peptides (2.5 μg in 50 μl of PBS) or active BoNT/A (1 μg in 50 μlPBS) were added to the wells of flexible polyvinyl chloride 96-wellplates (Becton Dickinson; San Jose, Calif.) and allowed to bind for 18hours at 4° C. After washing five times with PBS, the plates wereblocked for 1 hour at 37° C. with 1% bovine serum albumin (BSA) in PBS.Aliquots (50 μl) of anti-toxin antisera that had been prediluted with0.1% BSA in PBS (dilutions were human IgG fraction, 1:1000 and 1:2000(vol/vol)) were pipetted into the appropriate wells and kept at 4° C.for 20 hours. The wells were washed five times with PBS before adding 50μl of affinity-purified rabbit Ig against human IgG and IgM (DakoCorporation; Carpinteria, Calif.) diluted 1:1000 with 0.1% BSA in PBS tothe wells of the plate, and incubating for 2 hours at 37° C.

The wells were then washed five times with PBS, and 50 μl of¹²⁵I-labeled Protein A (2×10⁵ cpm in 0.1% BSA in PBS) was distributed tothe wells and allowed to incubate for 2 hours at room temperature.Finally, the plates were washed thoroughly to remove unboundradioactivity, the individual wells were cut out and transferred intoseparate tubes, and bound radioactivity was counted in a gamma-counter(1277 Gamma Master; LKB, Finland). Controls included binding ofpreimmune or normal sera to BoNT/A and its peptides, as well as bindingof immune sera to BSA and unrelated peptides. Assays were performed intriplicate. Results of the triplicate analyses were expressed as mean ofnet cpm A SD, after correction for nonspecific binding in control wellsthat were coated with BSA and unrelated peptides.

As shown in FIG. 2, human anti-BoNT antisera were observed to bind toseveral BoNT/A peptides. Peptide N25 (785-803) was observed to beimmunodominant followed, in decreasing order, by regions N8 (residues547-565 of SEQ ID NO:1), N22 (residues 743-761 of SEQ ID NO:1), and N16(residues 659-677 of SEQ ID NO:1). Lower, but reproducible, amounts ofantibodies were bound, in decreasing order, by peptides N11 (residues589-607 of SEQ ID NO:1), N17 (residues 673-691 of SEQ ID NO:1), N20(residues 715-733 of SEQ ID NO:1), N14 (residues 631-649 of SEQ IDNO:1), N28 (residues 827-845 of SEQ ID NO:1), N27 (residues 813-831 ofSEQ ID NO:1), N4 (residues 491-509 of SEQ ID NO:1), N24 (residues771-789 of SEQ ID NO:1) and N7 (residues 533-551 of SEQ ID NO:1). Theremaining H_(N) peptides bound little or no antibodies. As shown in FIG.2, human antibodies bound to the H_(C) peptides C2, C6, C10, C11, C15,C21, C24, C31 (FIG. 2) in agreement with previous studies (Atassi etal., supra, 1996). Human anti-BoNT antisera exhibited no binding to acontrol peptide corresponding to amino acids 218-231 of BoNT light chain(“L peptide). Nonimmune human IgG did not bind to any peptides, andhuman anti-BoNT antisera showed no antibody binding to unrelatedproteins and peptides. The results define antigenic portions of theH_(N) domain of BoNT/A.

The three-dimensional structure of BoNT/A reveals the solvent-exposedportions of the primary BoNT/A sequence (Lacy et al. Nat. Struct. Biol.5:898 (1996)). Comparison with the results obtained in the present studyrevealed that the immunodominant antibody-binding regions reside onsurface locations on the H subunit of BoNT/A.

In sum, these results demonstrate that BoNT/A peptides N25, N8, N22,N16, N11, N17, N20, N14, N28, N27, N4, N24, N7, C2, C6, C10, C11, C15,C21, C24, and C31 were recognized by human anti-BoNT antisera.

EXAMPLE II Mapping of Mouse Anti-Pentavalent Botulinum Toxoid AntibodiesUsing BoNT/A Synthetic Peptides

This example describes antigenic mapping of BoNT/A with mouse anti-BoNTantisera using 29 BoNT/A synthetic peptides that encompass the H_(N)domain of BoNT/A.

Mouse anti-BoNT antisera were prepared in outbred ICR mice bysubcutaneous immunization with BoNT pentavalent toxoid. Antisera used inthese studies were obtained 91 days after the first injection (Atassi etal., supra, 1996). Mice were purchased from the National CancerInstitute, and Jackson Laboratory (Bar Harbor, Me.). For use ascontrols, non-immune mouse sera were obtained from the animals beforeimmunization.

Peptide binding assays were performed as described in Example I, exceptthat the dilution for antisera of outbred mice was 1:50 and 1:200(vol/vol). The secondary antibodies (mouse IgG (H+L)+IgM (Mu chain) wereobtained from Accurate Chemical & Scientific Corporation (Westbury, NewYork) and were diluted 1:2000 (vol/vol).

As shown in FIG. 3, mouse anti-BoNT antisera were observed to bind toseveral BoNT/A peptides. At a dilution of 1:50 (vol/vol), peptide N25(785-803) was immunodominant, followed by one or more regions within theoverlap N6/N7/N8/N9 (residues 519-537/533-551/547-565/561-579 of SEQ IDNO:1) and one or more weaker regions within the overlap N27/N28(residues 813-831/827-845 of SEQ ID NO:1). At a dilution of 1:200(vol/vol), peptide N25 (residues 785-803 of SEQ ID NO:1) remainedimmunodominant; in addition, high amounts of antibodies were bound bythe overlap N6/N7/N8 (residues 519-537/533-551/547-565 of SEQ ID NO:1),low amounts of antibodies were bound by the overlap N27/N28 (residues813-831/827-845 of SEQ ID NO:1), indicating that at least one weakepitope resides within this region (See FIG. 3). As shown in FIG. 3, theH_(C) peptides that possessed antibody binding were C2, C7, C11, C15,C16, C24 and C31, in agreement with previously reported results (Atassiet al., supra, 1996). Mouse anti-BoNT antisera exhibited no binding to acontrol peptide corresponding to amino acids 218-231 of BoNT light chain(“L peptide”). The mouse anti-BoNT antisera exhibited no antibodybinding to unrelated proteins and peptides. Preimmune sera from the samemice did not bind to any of the H_(N) or H_(C) peptides.

In sum, these results demonstrate that peptides N25, N6, N7, N8, N9,N27, N28, C2, C7, C11, C15, C16, C24 and C31 were recognized by mouseanti-BoNT antisera.

EXAMPLE III Mapping of Chicken BoNT/A Toxoid Antibodies Using BoNT/ASynthetic Peptides

This example describes antigenic mapping of BoNT/A with chickenanti-BoNT antisera using 29 BoNT/A synthetic peptides that encompass theH_(N) domain of BoNT/A

Chicken antisera were prepared by monthly subcutaneous injection offormaldehyde-inactivated BoNT/A in Ribi adjuvant. Sera used in thisstudy were obtained after four injections. For use as controls,non-immune chicken sera were obtained from the animals beforeimmunization.

Peptide binding assays were performed as described in Example I, exceptthat the dilution for chicken antisera was 1:500 (vol/vol). Thesecondary antibodies (rabbit antiserum against chicken IgG) were diluted1:500 (vol/vol).

TABLE 1 Sequence Position Peptide (residues of No. SEQ ID NO: 1) HumanHorse Mouse Chicken C1 855-873 − +++ ± − C2 869-887 +++ − +++ +++ C3883-901 − + + +++++ C4 897-915 − ± − ± C5 911-929 ++ + − + C6 925-943+++ − − ++ C7 939-957 + ++ + +++++ C8 953-971 − − − − C9 967-985 + − ±++++ C10 981-999 +++ ± − +++++ C11  995-1013 +++++ + + +++++ C121009-1027 − − − + C13 1023-1041 − + − − C14 1037-1055 − + − + C151051-1069 +++++ ± ++ +++++ C16 1065-1083 − − − − C17 1079-1097 − + − −C18 1093-1111 − + + ++ C19 1107-1125 ± − − − C20 1121-1139 + + ± +++++C21 1135-1153 ++ ± ± +++ C22 1149-1167 ± + − ++ C23 1163-1181 ± − − −C24 1177-1195 +++ − ++ +++++ C25 1191-1209 ± ++ − − C26 1205-1223 − + −− C27 1219-1237 + − − − C28 1233-1251 + − − − C29 1247-1265 ++ ± − ± C301261-1279 + ++ − +++ C31 1275-1296 ++ +++ ++ +++ Active +++++ ++++++++++ +++++ BoNT/A (+) or (−) signs are based on net cpm values anddenote the following: (−), less than 1,500 cpm; (Å), 1,500-3,000 cpm;(+), 3,000-7,000 cpm; (++), 7,000-15,000 cpm; (+++), 15,000-25,000 cpm;(++++), 25,000-35,000 cpm; (+++++), exceeding 35,000 cpm.

As shown in FIG. 4, chicken anti-BoNT antisera were observed to bind toseveral BoNT/A peptides. In particular, peptide N25 (residues 785-803 ofSEQ ID NO:1) was the most immunodominant region, followed by N8(residues 547-565 of SEQ ID NO:1) (FIG. 4). In addition, lower levels ofantibodies were directed, in the following decreasing order of antibodylevel, against peptides N22 (residues 743-761 of SEQ ID NO:1), N27(residues 813-831 of SEQ ID NO:1), N28 (residues 827-845 of SEQ IDNO:1), N7 (residues 533-551 of SEQ ID NO:1), N6 (residues 519-537 of SEQID NO:1), N19 (residues 701-719 of SEQ ID NO:1) and N20 (residues715-733 of SEQ ID NO:1). The antibody-binding profile of the peptidescorresponding to the entire H chain, including the H_(C) domain is shownin FIG. 4. In the H_(C) domain, chicken antibodies recognizedessentially seven major regions, each of which can contain one or moreantigenic sites or epitopes. The regions were located within thepeptides C15 (residues 1051-1069 of SEQ ID NO:1) and C24 (1177-1195 ofSEQ ID NO:1) and the overlaps C2/C3 (residues 869-887/883-901 of SEQ IDNO:1), C6/C7 (residues 925-943/939-957 of SEQ ID NO:1), C9/C10/C11(residues 967-985/981-999/995-1013 of SEQ ID NO:1), C20/C21/C22(residues 1121-1139/1135-1153/1149-1167 of SEQ ID NO:1) and C30/C31(residues 1261-1279/1275-1296 of SEQ ID NO:1). The chicken antiserashowed no antibody binding to unrelated proteins and peptides, andchicken anti-BoNT antisera exhibited no binding to a control peptidecorresponding to amino acids 218-231 of BoNT light chain. Preimmunechicken sera bound none of the H_(N) or H_(C) peptides. The bindingprofile of the chicken anti-BoNT/A antibodies to the panel of H_(C)peptides was similar to that of human antibodies as shown in Table 1.

In sum, these results demonstrate that peptides N25, N8 N22, N27, N28,N7, N6, N19, N20, C15, C24, C2, C3, C6, C7, C9, C10, C11, C20, C21, C22,C30, and C31 were recognized by chicken anti-BoNT antisera.

EXAMPLE IV Mapping of Horse BoNT/A Toxoid Antibodies Using BoNT/ASynthetic Peptides

This example describes antigenic mapping of BoNT/A with horse anti-BoNTantisera using 29 BoNT/A synthetic peptides that encompass the H_(N)domain of BoNT/A.

Horse antisera were prepared by subcutaneous immunization, in multiplesites every two weeks for over a year, with a formaldehyde-inactivatedBoNT/A in Ribi adjuvant. The antisera tested in the binding studies wereobtained after four injections according to procedures described inAtassi et al., supra, 1996. For use as controls, non-immune horse serawere obtained from the animals before immunization.

Peptide binding assays were performed as described in Example I, exceptthat the dilution for horse antisera was 1:300 (vol/vol). The secondaryantibodies were affinity purified rabbit anti-horse IgG obtained fromAccurate Chemical & Scientific Corporation (Weston, New York) and werediluted 1:500 (vol/vol).

As with the antisera of human, mouse and chicken as described inExamples I, II and III, one or more regions within the overlappingpeptides N7/N8/N9 (residues 533-551/547-565/561-579 of SEQ ID NO:1) wereobserved to be immunodominant, and peptides N27 (residues 813-831 of SEQID NO:1), N25 (residues 785-803 of SEQ ID NO:1), N22 (residues 743-761of SEQ ID NO:1) and N20 (residues 715-733 of SEQ ID NO:1) possessedbinding activity (see FIG. 4). However, horse antibodies exhibited ahigh level of binding to peptide N2 (residues 463-481 of SEQ ID NO:1),whereas other sera had low levels of binding to peptide N1 (residues449-467 of SEQ ID NO:1). Therefore, the horse immune response to theBoNT/A region in the vicinity of peptide N2 is shifted to the right by afew residues. The N2 region is also more immunogenic in horse than inhuman, mouse and chicken. As shown in FIG. 5, horse anti-BoNT antiserawere also observed to bind to H_(C) peptides C1, C5, C7, C18, C22, C25,C30 and C31, in agreement with previous studies (Atassi et al., supra,1996). Using the horse anti-BoNT antisera, no binding to a controlpeptide corresponding to amino acids 218-231 of BoNT light chain wasobserved. The antisera had no binding to unrelated proteins, andpreimmune horse sera bound none of the H_(N) or H_(C) peptides.

In sum, these results demonstrate that peptides N7, N8, N9, N27, N25,N22, N20, N2, N1, C1, C5, C7, C18, C22, C25, C30 and C31 were recognizedby horse anti-BoNT antisera.

EXAMPLE V Comparison of BoNT/A Antigenicity Between Human, Mouse,Chicken and Horse

This example defines several common immunogenic regions of BoNT/A byantigen mapping obtained with antisera from four different species.

The results shown in Examples I through IV indicate that antiseraagainst BoNT/A raised in human, horse, mouse and chicken recognizesimilar immunodominant regions on the H_(N) domain of BoNT/A. Theseregions resided, with slight shifts to the left or to the right, withinthe peptides N6/N7/N8/N9 (residues 519-537/533-551/547-565/561-579 ofSEQ ID NO:1) overlap (human, horse and mouse), peptide N22 (residues743-761 of SEQ ID NO:1) (human, horse and chicken), peptide N25(residues 785-803 of SEQ ID NO:1) and peptides N27/N28 (residues813-831/827-845 of SEQ ID NO:1). These results are summarized in Table2, below.

TABLE 2 Sequence Position (residues of Peptide No. SEQ ID NO: 1) HumanHorse Mouse Chicken L-Peptide 218-231 − − − − N1 449-467 ++ − + ± N2463-481 − +++++ − − N3 477-495 − ± − − N4 491-509 ++ + ± + N5 505-523− + − − N6 519-537 ++ + +++ ++ N7 533-551 ++ +++ +++ +++ N8 547-565+++++ +++++ +++++ +++++ N9 561-579 + ++++ ++++ ± N10 575-593 ± ++ + ++N11 589-607 +++ + − + N12 603-621 + − − − N13 617-635 − ± − − N14631-649 ++ ± ± + N15 645-663 − − − ± N16 659-677 ++++ − − − N17 673-691++ − ± ++ N18 687-705 + ± − − N19 701-719 ± + + ++ N20 715-733 ++ ++ ±++ N21 729-747 ± − − − N22 743-761 ++++ ++ + ++++ N23 757-775 − + − −N24 771-789 ++ ± + + N25 785-803 +++++ +++ +++++ +++++ N26 799-817 − − −− N27 813-831 ++ ++++ +++ ++++ N28 827-845 ++ + +++ +++ N29 841-859 + +− − Active +++++ +++++ +++++ +++++ BoNT/A (+) or (−) signs are based onnet cpm values and denote the following: (−), less than 1,500 cpm; (Å),1,500-3,000 cpm; (+), 3,000-7,000 cpm; (++), 7,000-15,000 cpm; (+++),15,000-25,000 cpm; (++++), 25,000-35,000 cpm; (+++++), exceeding 35,000cpm.

Whereas peptide N2 was strongly immunodominant with horse antisera, itwas unreactive with human, mouse and chicken antisera. However withhuman, mouse and chicken antisera, peptide N1 reacted weakly andtherefore, the reaction of horse antibodies with peptide N2 canrepresent a shift to the right of the epitope recognized by the horseantibodies. The overlap N16/N17 was highly reactive with humanantibodies, whereas with mouse and chicken antisera peptide 17 showed alow level of reactivity. With horse antisera, antibodies against N16/N17were not detected.

In sum, this example shows that anti-BoNT antibodies from human, mouse,horse and chicken recognize several common immunogenic regions of theBoNT/A H_(N) domain.

EXAMPLE VI Identification of Immunodominant Regions of BoNT/A

This example shows the identification of several immunodominant regionsof human anti-BoNT antibodies within the H chain of BoNT/A.

The antigenic regions of BoNT were determined using anti-BoNT antiseraobtained from human, mouse, horse and chicken, as shown in Examples Ithrough IV. The location of antigenic regions can be narrowed to shorterdomains by the following analysis.

In this analysis, the size of an antigenic site was assigned to be 10-11residues. The H-chain of BoNT/A was therefore broken down into 13antigenic sites. The 13 antigenic sites are defined in Table 3, below.The table gives the approximate locations of only the antigenic regionsthat bind 15,000 cpm of antibody or greater. Although only theimmunodominant regions are shown in Table 3, regions binding loweramounts of antibodies can be of equivalent immunological significance.

In sum, this example shows that BoNT/A immunodominant regions having10-11 residues can be determined based on reactivity of anti-BoNTantisera obtained from human, mouse, horse and chicken with BoNT/Apeptides.

TABLE 3 Antigenic Regions Amino Acid Residue of SEQ ID NO: 1 H_(N)Domain Regions NR1 554-564 NR2 593-602 NR3 666-676 NR4 748-757 NR5785-794 H_(C) Domain Regions CR1 854-887 CR2 933-943 CR3 986-995 CR41000-1009 CR5 1056-1065 CR6 1137-1147 CR7 1183-1192 CR8 1276-1289

EXAMPLE VII Mapping of T- and B-Cell Recognition Profiles of the BoNT/AH_(N) Domain in Two High-Responder Mouse Strains

This example demonstrates that responses to each antibody or T cellepitope are under separate genetic control and that there is partial,but not complete, coincidence between antibody and T cell H_(N)recognition regions.

A. T Cell Recognition of H_(N) Peptides After One Injection with Toxoid

Exemplary proliferative responses of BALB/c lymph node cells (LNCs) weredetermined at various doses of toxoid as shown in FIG. 7. The responseprofile to the full panel of H_(N) peptides spanning the entireN-terminal domain of the BoNT/A heavy chain was subsequently determined.As shown in FIG. 8, BALB/c T cells primed with one injection of BoNT/Atoxoid recognized one major region localized within overlap N18/N19(residues 687-705/701-719 of SEQ ID NO: 1) while the remaining peptideshad no detectable stimulating activity in vitro. BoNT/A-primed BALB/c Tcells showed substantial cross-reaction with BoNT/B (SI values: BoNT/A23.62, BoNT/B 7.89) but had no cross-reactivity with TeNT (FIG. 7).

Unlike BALB/c T cells, the T cells from a BoNT/A-primed second inbredstrain of mice, SJL/JCr, cross-reacted with both BoNT/B and TeNT (FIG.9). As summarized in FIG. 10, BoNT/A-primed SJL T cells responded tochallenge with a number of the overlapping peptides of H_(N). Inparticular, peptides N9 (residues 561-579 of SEQ ID NO: 1), N11(residues 589-607 of SEQ ID NO: 1), N13 (residues 617-635 of SEQ ID NO:1), N29 (residues 841-859 of SEQ ID NO: 1) and the L-chain peptide(218-231) stimulated strong-to-medium in vitro T cell responses (SI>5).In addition, peptides N2 (residues 463-481 of SEQ ID NO: 1), N16(residues 659-677 of SEQ ID NO: 1) and N21 (residues 729-747 of SEQ IDNO: 1) and N28 (residues 827-845 of SEQ ID NO: 1) demonstrated weak(SI>3) stimulating activities. Toxoid-primed T cells of BALB/c and SJLdid not respond to the unrelated hen lysozyme or ovalbumin proteins,demonstrating the specificity of the response.

Female BALB/c (H-2^(d); National Cancer Institute; Frederick, Md.) andSJL/JCr (H-2^(s); (Jackson Laboratory; Bar Harbor, Me.) mice, 7 to 9weeks old, were used in all experiments. Synthetic peptides weresynthesized, purified and characterized as described above. Thetwenty-nine consecutive overlapping peptides correspond to the completeH_(N) domain (residues 449-859 of SEQ ID NO: 1) and a peptide around theenzymatic active site of the light chain (L-peptide, residues 218-231)of BoNT/A (FIG. 1A). The peptides were 19 residues in length andoverlapped consecutively by five residues.

Immunization of mice with BoNT/A toxoid for T cell studies was performedas follows. The optimum priming dose of BoNT/A toxoid was determined inthe BALB/c and SJL mouse strains. Mice were immunized subcutaneously atthe base of tail with various doses of toxoid (0.125-5 μg/mouse) in a50-μl emulsion of equal volumes of the toxoid solution in 0.15 M NaCl in0.01 M sodium phosphate buffer, pH 7.2 (PBS), and complete Freund'sadjuvant (CFA) containing Mycobacterium tuberculosis, strain H37Ra(Difco Laboratories; Detroit, Mich.). For both mouse strains, thehighest T cell response was obtained at a priming dose of 1 μg/mouse,and subsequent experiments were performed with this dose. The peptideswere used in vitro at five doses (5, 10, 20, 40, 80 μg/ml), and thetoxin was used in vitro at doses of 1.25, 2.5, 5 and 10 μg/ml.

Lymphocyte proliferation assays were performed as follows. Single-cellsuspensions of LNC from toxoid-primed mice were prepared in Hank'sbalanced salt solution. The cells were washed and resuspended in RPMI1640 with 1% normal mouse serum and supplemented as described inRosenberg et al., Immunol. Invest. 26:491-504 (1997). The number ofviable cells was determined by vital staining with fluoresceindiacetate. A fixed number of viable LNC (5×10⁵ to 8×10⁵ cells/well) wascocultured in triplicate with various concentrations of mitogen, BoNT/Aor synthetic peptides of BoNT/A, BoNT/B or TeNT and control proteins andpeptides. The viability of the cells was confirmed in each assay bytheir responses to ConA and LPS. Negative controls included proteinsunrelated to BoNT/A (ovalbumin, myoglobin and hen lysozyme) as well asunrelated control synthetic peptides. After three days of incubation at37° C. in a humidified, 5% CO₂ atmosphere, lymphocytes were pulsed for18 hours with [³H]-thymidine (2 μCi/well; Research ProductsInternational; Mount Prospect, Ill.) and subsequently harvested ontoglass microfiber filters (Whatman; Clinton, N.J.) before counting byliquid scintillation.

B. Mapping of the T Cell Recognition Profiles After Three Injectionswith Toxoid

To determine T cell recognition profiles at the time antisera wereobtained, proliferative responses were determined for LNC obtained fromBALB/c and SJL mice that were used to prepare hyperimmune anti-toxoidantisera for the antibody-binding studies. LNC were harvested at thetime of the final bleed on week 10 (i.e. 2 weeks after the last of threeinjections of toxoid). The proliferative responses to the peptides andtoxins of LNC from once-primed and from three-times immunized BALB/c andSJL are shown in FIGS. 8 and 10; the results for both BALB/c and SJL aresummarized in Table 4. As shown in FIG. 8, the two recognition profilesfor T cells from BALB/c mice were only slightly different (FIG. 8).Hyperimmune T cells responded to challenge in vitro with peptides N18(residues 687-705 of SEQ ID NO: 1), N19 (residues 701-719 of SEQ IDNO: 1) and N20 (residues 715-733 of SEQ ID NO: 1), with the response topeptide N19 (residues 701-719 of SEQ ID NO: 1) stronger after multipleinjections. The recognition profile of the other peptides remainedessentially unchanged, and BALB/c hyperimmune T cells did notcross-react with BoNT/B and TeNT.

The recognition profiles of once-primed and of hyperimmune LNC from SJLmice showed greater differences (FIG. 10 and Table 4). As shown in FIG.10, hyperimmune T cells showed higher cross-reactivity with BoNT/B andTeNT than once-primed cells. In addition, the responses of hyperimmuneSJL T cells to peptides N2 (residues 463-481 of SEQ ID NO: 1), N9(residues 561-579 of SEQ ID NO: 1), N13 (residues 617-635 of SEQ ID NO:1), N22 (residues 743-761 of SEQ ID NO: 1) and N29 (residues 841-859 ofSEQ ID NO: 1) increased markedly. Hyperimmune SJL T cells also respondedwell to peptides N3 (residues 477-495 of SEQ ID NO: 1), N5 (residues505-523 of SEQ ID NO: 1), N6 (residues 519-537 of SEQ ID NO: 1), N7(residues 533-551 of SEQ ID NO: 1), N8 (residues 547-565 of SEQ ID NO:1), N10 (residues 575-593 of SEQ ID NO: 1), N11 (residues 589-607 of SEQID NO: 1), N24 (residues 771-789 of SEQ ID NO: 1), N26 (residues 799-817of SEQ ID NO: 1), N27 (residues 813-831 of SEQ ID NO: 1) and theL-peptide (218-231).

TABLE 4 The regions on the H chain of BoNT/A recognized by T cells afterone injection and after 3 injections of BALB/c and SJL with BoNTAtoxoid. Residue Number BALB/c (H-2^(d)) SJL (H-2⁵) (SEQ 1 3 1 3 PeptideID NO: 1) Injection Injections Injection Injections L-Peptide 218-231 −− ++ ++ H_(N) Domain^(a) N1 449-467 − − − − N2 463-481 − − + ++++ N3477-495 − − − ++ N4 491-509 − − − − N5 505-523 − − ± ++ N6 519-537 − − −++ N7 533-551 − − − ++ N8 547-565 − − − + N9 561-579 − − +++ ++++ N10575-593 − − ± + N11 589-607 − − ++ + N12 603-621 − − − − N13 617-635 − −+++++ +++++ N14 631-649 − − − − N15 645-663 − − − − N16 659-677 − − + +N17 673-691 − − − − N18 687-705 ± ± − ± N19 701-719 + ++ − − N20 715-733− + − − N21 729-747 − − + − N22 743-761 − − − ++++ N23 757-775 − − − ±N24 771-789 − − − + N25 785-803 − − − − N26 799-817 − − − ++ N27 813-831− − − + N28 827-845 − − + ± N29 841-859 − − ++ +++ BoNT/A +++++ ++++++++++ +++++ NoNT/B + +++ +++++ +++++ TeNT − − +++++ +++++

Immunization of mice with BoNT/A toxoid for late T cell responses andantibody binding studies was performed as follows. Mouse antisera wereprepared by injection of BALB/c and SJL mice subcutaneously in the hindfootpads with 5 μg of toxoid emulsified in complete Freund's adjuvant(CFA). Mice were injected with boosters at 4 and 8 weeks with a similardose of toxoid, using incomplete Freund's adjuvant (Difco Laboratories;Detroit, Mich.) instead of CFA. Sera were collected prior to the firstimmunization (pre-immune sera) and two weeks after each injection. Foreach mouse strain, sera of the respective bleeds from ten mice werepooled and kept at −20° C. until use. Antisera collected on week 10,i.e. 2 weeks after the last injection with toxoid, were employed forpeptide binding studies. At the time of the last bleed, lymph nodes wereremoved, and single cell suspensions prepared for lymphocyteproliferation assays.

C. Binding of Anti-BoNT/A Antibodies to Overlapping Synthetic Peptidesand Toxins

Mapping of antibody binding profiles to peptides in the BALB/c and SLJinbred mouse strains was performed by assaying antisera at two dilutions(1:250 and 1:500 (vol/vol)). As shown in FIGS. 5 and 6, respectively,the binding profiles of anti-toxoid antibodies from BALB/c and SJL micewere substantially similar. FIG. 13 shows a direct comparison of BALB/cand SJL antisera binding, and Table 5 summarizes the binding profilesfor BALB/c and SJL Abs to the H_(N) peptides at a dilution of 1:250(vol/vol). Antibodies from both mouse strains showed high binding toH_(N) peptides N7, N8, N25 and N27 and low binding to peptides N6, N11,N15 and N19.

TABLE 5 The regions on the H chain of BoNT/A recognized by T cellsand/or Abs after 3 injections of BALB/c and SJL with BoNT/A toxoid.Residue Number BALB (H-2^(d)) SJL (H-2⁵) Peptide (SEQ ID NO: 1) AbsT-cells Abs T-cells L-Peptide 218-231 + − + ++ H_(N) Domain^(a) N1449-467 ± − − − N2 463-481 − − − ++++ N3 477-495 − − − ++ N4 491-509 ± −− − N5 505-523 − − − ++ N6 519-537 + − + ++ N7 533-551 +++ − +++ ++ N8547-565 ++++ − ++++ + N9 561-579 ± − +++ ++++ N10 575-593 + − − + N11589-607 + − − + N12 603-621 − − − − N13 617-635 − − − +++++ N14 631-649− − − − N15 645-663 + − + − N16 659-677 − − − + N17 673-691 − − − − N18687-705 − ± − ± N19 701-719 + ++ + − N20 715-733 + + − − N21 729-747 − −− − N22 743-761 ± − +++ ++++ N23 757-775 − − − ± N24 771-789 + − − + N25785-803 ++++ − ++++ − N26 799-817 − − − ++ N27 813-831 +++ − ++++ + N28827-845 ++ − ± ± N29 841-859 − − − +++ BoNT/A +++++ +++++ NoNT/B +++++++ TeNT − +++++ H_(C) Domain^(b) C1 855-873 − − + − C2 869-887 ++ −+++ − C3 883-901 ++ − ++ − C4 897-915 − ++ − ++++ C5 911-929 − − + + C6925-943 + − + + C7 939-957 + ++ + +++ C8 953-971 − − − ++ C9 967-985 +− + − C10 981-999 + − + − C11  995-1013 + − +++ − C12 1009-1027 − + − +C13 1023-1041 + − − ++ C14 1037-1055 − − − + C15 1051-1069 + − ++ +++C16 1065-1083 − − − + C17 1079-1097 − − − ++ C18 1093-1111 − ± + + C191107-1125 + ++ + + C20 1121-1139 − + + ++ C21 1135-1153 ++ − + + C221149-1167 − − + + C23 1163-1181 − − − ++ C24 1177-1195 − − +++ + C251191-1209 − − + + C26 1205-1223 − − + − C27 1219-1237 − − − − C281233-1251 − − + + C29 1247-1265 − − − + C30 1261-1279 − + − C311275-1296 ++ − ++ ++ ^(a)Results from the present work. For the purposeof this table, (+) and (−) assignments were based on net cpm values forAb binding and SI values for T cell proliferation. For Ab binding, thesymbols denote the following values: (−), less than 1,500 cpm; (±),1,500-3,000 cpm; (+), 3,000-7,000 cpm; (++), 7,000-15,000 cpm; (+++),15,000-25,000 cpm; (++++), 25,000-35,000 cpm; (+++++), exceeding 35,000cpm. For T cell proliferation, the symbols indicate the following: (−),SI value less then 2.0; (±) 2.0-2.5; (+), SI 2.6-3.5; (++), SI 3.6-6.0;(+++), SI 6.1-10.0; (++++), 10.1-25; (+++++), SI > 25.0. ^(b)Results ofThe Hc domain peptide recognition by anti-toxoid Abs and T cells ofBALB/c and SJL mice are from Rosenberg et al., 1997.

Some differences in the binding profiles of antibodies from the twomouse strains were also apparent. In particular, BALB/c antisera showedmedium antibody binding to peptide N28 and low antibody binding topeptides N10, N20 and N24, which represented epitopes eitherunrecognized or poorly recognized by SJL antibodies. On the other hand,SJL antibodies showed high binding to peptides N9 and N22, which werepoorly recognized by BALB/c antibodies. In addition, SJL antiseracontained much higher amounts of antisera that bound to peptide N27 thandid antisera from the other mouse strain. In order to complete theprofiles of the H chain recognition by BALB/c and SJL antibodies, Table5 shows binding profiles to H_(C) peptides previously reported(Rosenberg et al., supra, 1997).

Solid phase radioimmunoassays were performed using Staphylococcalprotein A (Pharmacia Biotech; Piscataway, N.J.) radiolabeled with ¹²⁵I(Amersham Corp.; Arlington Heights, Ill.) using the chloramine-T method.Unbound ¹²⁵I was separated from the radiolabeled protein A by gelfiltration on a column (0.8×20 cm) of Sephadex G-25, equilibrated withPBS containing 0.1% bovine serum albumin (BSA; Sigma Chemicals; St.Louis, Mo.).

Binding of mouse anti-toxoid antibodies to active BoNT/A and tosynthetic peptides was determined using polyvinylchloride 96-well plates(Becton Dickinson Labware; Oxnard, Calif.), which were coated with eachof the 31 overlapping peptides (2.5 μg in 50 μl of PBS/well) or withBoNT/A (1 μg in 50 μl of PBS/well). Wells coated with proteins andsynthetic peptides unrelated to BoNTs were used as negative controls.Following overnight incubation at 4° C., plates were washed extensivelywith PBS and incubated for one hour at 37° C. with 1% BSA in PBS (100μl/well) to block nonspecific binding in subsequent steps. After washingwith PBS, plates were incubated at 37° C. for three hours with mouseantisera (50 μl/well) appropriately prediluted in 0.1% BSA in PBS. Formapping studies, antisera were prediluted 1:250 and 1:500 (vol/vol).Wells were washed with PBS and incubated at 37° C. for two hours with 50μl of affinity purified rabbit anti-mouse (IgG+IgM) antisera (AccurateChem. Sci. Corp.; Westbury, N.Y.) pre-diluted 1:1000 (vol/vol) with 0.1%BSA in PBS. After washing with PBS, ¹²⁵I-labeled protein A was added tothe wells (2×10⁵ cpm in 50 μl 0.1% BSA-PBS/well). Plates weresubsequently incubated for two hours at room temperature, washed, driedand the wells cut out and counted in a gamma counter (1227 Gammamaster;LKB; Turku, Finland). All determinations were performed in triplicate,and the results expressed as net cpm ±SD, after corrections fornonspecific binding in controls wells that were coated with BSA and ofthe correlate pre-immune mouse sera to each tested antigen.

D. Protective Activity of Anti-BoNT/A Antibodies In Vivo

Anti-BoNT/A antisera from BALB/c and SJL mice were assayed for theability to protect against a lethal dose of active BoNT/A as describedfurther below. Serial dilutions of BALB/c and SJL antisera were assayedfor the ability to protect ICR mice against 1.05×LD₁₀₀ (i.e., 6.5 pg) ofBoNT/A. As shown in FIG. 14, antisera of both BALB/c and SJL containedhigh titers of blocking antibodies that protected mice at very highdilutions. Anti-toxin antisera of BALB/c mice were fully protective inrecipient ICR mice at dilutions up to 1:28000 (vol/vol), and 50%protection was obtained at 1:38000 (vol/vol). SJL antisera were evenmore protective, fully protecting recipient ICR mice against a lethaldose of active BoNT/A at 1:36000 dilution (vol/vol), while 50%protection was achieved at 1:41000 dilution (vol/vol). As expected,non-immune sera were not protective at any dilution. These resultsindicate that anti-toxoid antibodies can be useful for conferringprotection against botulinum toxin.

The presence of blocking antibodies in mouse antisera against BoNT/A wasdetermined by a mouse protection assay essentially as follows. Thesurvival of outbred (ICR) mice against various doses of BoNT/Aadministered intravenously was determined using five mice at each dose.The dose at which no mice survived (i.e., LD₁₀₀) was 5.0 pg/mouse when afresh preparation of BoNT/A was tested. At the time the mouse protectionassays were performed, after storage of toxoid for about 6 months at−20° C. in PBS containing 20% glycerin, the LD₁₀₀ was 6.2 pg/mouse. Todetermine the protective activity of BALB/c and SJL anti-BoNT/Aantisera, ICR mice were injected intravenously in the tail with amixture of 1.05×LD₁₀₀ of active BoNT/A (i.e., 6.5 pg/mouse) and 100 μlof serial dilutions of the indicated mouse antiserum. Each dilution wasinjected into five mice, and the mice were observed three times a dayfor six days. Where test antisera contained blocking antibodies, allmice recovered and survived the challenge. When protecting antibodieswere either absent or their amounts too low at high dilution, then noneor only some of the mice survived the BoNT/A challenge. The results wereplotted as percent survival versus antisera dilutions.

EXAMPLE VIII Submolecular Recognition Profiles in Two Mouse StrainsOfnon-Protective and Protective Anti-BoNT/A Antibodies

This example demonstrates that the switch in BALB/c and SJL mice fromnon-protective to protective antibodies is not associated with majorchanges in epitope recognition profiles but is rather associated withthe immunoglobulin class of the antibodies.

A. Protective Activity of Anti-BoNT/A Antibodies In Vivo

As described above, female BALB/c (H-2^(d)) and SJL/JCr (H-2^(s)) mice,7 to 9 weeks old, were used in all experiments. The mouse protectionassay was performed as described in Example VII above.Formaldehyde-inactivated, and active BoNT/A were purchased fromMetabiologics (Madison, Wis.).

Anti-toxin antisera of BALB/c and SJL remained unprotective in recipientICR mice on 26 day after the first BoNT/A injection. Mice were boostedon day 27, and nine days after the second injection (i.e., day 36 afterthe first injection), antisera were tested for protection. BALB/cantisera were protective against a challenge dose of 1.05×LD₁₀₀, whenadministered undiluted. SJL antisera were protective on day 36; theseantisera were protective at dilutions up to 1:4 and were not protectiveat dilutions of 1:8. Non-immune sera were not protective even whenundiluted. These results serve to define the timing of the switchbetween production of unprotective and protective anti-BoNT/Aantibodies.

B. Binding Profile of Non-Protective and Protective Total Antibodies

For mapping of peptide binding profiles, antisera were assayed atdilutions of 1:100 and 1:250 (vol/vol). Binding profiles of total (IgGand IgM) anti-toxin antibodies from BALB/c and SJL mice were determinedfor two bleeds: The bleed on day 26 containing non-protective antibodiesand the bleed following it on day 36 in which the antibodiesdemonstrated protective activity.

As shown in FIG. 16, upper panel, non-protective and protective BALB/cantisera showed very similar peptide-binding profiles at a dilution of1:100. At a dilution of 1:250, the protective BALB/c antisera displayedhigher binding to essentially every peptide (FIG. 16, lower panel). TheBALB/c antibody-binding peptides were: N6/N7, N25, C2/C3, C9/C10/C11,C15, C18, C24, C30 and C31. Antibodies in the non-protective andprotective antisera bound to peptide C30 at similar levels at a dilutionof 1:100. However, at a dilution of 1:250, antibody binding to C30 inthe non-protective antisera was greatly diminished while binding in theprotective antisera remained unaffected, indicating a lower affinity ofthe antibodies directed against region C30 in the non-protectiveantisera. Low, but reproducible amounts of antibodies were bound bypeptides N19, C6/C7 and C28.

The binding profiles for SJL total antibodies are shown in FIG. 17 atdilutions of 1:100 and 1:250 (vol/vol), upper and lower panels,respectively. In the case of the SJL mice, some differences wereapparent between non-protective and protective antisera when totalantibodies were analyzed. Peptides N5, N22 and C21, which wererecognized by protective antisera, were only slightly recognized (N22and C21) or not recognized (N5) by non-protective sera. Additionally, inthe protective antisera, peptides N7/N8, N25, C11, C15 and less soN27/N28 bound two-fold or higher amounts of antibodies as compared withnon-protective antisera. Additional peptides, C4 and C29, bound higheramounts of antibodies in protective sera as compared to non-protectivesera at a dilution of 1:100. However these differences disappeared at1:250, indicating that these antibodies were of relatively low affinity.Peptides C2/C3, C7, C18/C19, C24, C30 and C31 also bound higher amountsof antibodies in protective sera as compared with non-protectiveantisera, but the differences were less than double. As expected,anti-toxin antibodies did not bind to unrelated proteins or peptides,and pre-immune sera displayed no binding to BoNT/A or its peptides,indicative of specific binding.

In sum, these results demonstrate only very small differences betweenthe peptide recognition profiles of protective and non-protectiveantisera. These results further indicate that differences in antibodybinding levels likely do not account for the difference in protectiveactivity of the non-protective and protective antisera.

Assays were performed as follows. A total of 60 consecutive overlappingpeptides corresponding to the complete H subunit (residues 449-1296 ofSEQ ID NO: 1), and a peptide around the enzymatic active site of thelight chain (L-peptide, residues 218-231), of BoNT/A (FIG. 1) weresynthesized, purified and characterized as described above. The peptideswere 19 residues long and overlapped consecutively by five residuesexcept for the last peptide in the sequence (C31, residues 1275-1296 ofSEQ ID NO: 1). Mice were immunized as described above, with two boostersgiven at days 27 and 60 with a similar dose of toxoid, using incompleteFreund's adjuvant. Sera were collected prior to the first immunization(preimmune sera) and on days 20, 26, 36, 46, 57, 68 and 70. For eachmouse strain, sera of the respective bleeds from 10 mice were pooled andkept at −20° C. The non-protective sera from day 26 and protective serafrom day 36 were employed for peptide binding studies. Binding wasdetermined by solid-phase radioimmunoassay as described in Example VIIabove, except that affinity-purified rabbit anti-mouse (IgG and IgM) oranti-mouse IgG antisera (Accurate Chem. Sci. Corp.; Westbury, N.Y.) wasused as appropriate.

C. Binding of Non-Protective and Protective IgG Antibodies to SyntheticBoNT/A Peptides and to BoNT/A

As described above, differences in total antibody reactivity betweenprotective and non-protective antisera, particularly in the case ofBALB/c antisera, appeared insufficient to explain the protectiveproperties of the antisera. The peptide-binding profiles of IgGantibodies alone showed different results. In their binding to activeBoNT/A, BALB/c and SJL protective antisera had 13-36 fold higher levelsof IgG antibodies relative to non-protective antisera. The profiles forBALB/c and SJL protective and non-protective antibodies are shown inFIGS. 18 and 19, respectively. IgG antibodies in the protective antiseraof each mouse strain bound to the same peptides as did total antibodies(IgG and IgM) in the correlate antiserum. However, in both mousestrains, the non-protective antisera contained few, if any, IgGantibodies that bound to these peptides, even at a dilution of 1:100.Again, specific binding was demonstrated by the absence of binding tounrelated proteins and peptides, and by the absence of BoNT/A binding bynon-immune sera.

These results demonstrate that protective antibodies had much higher IgGlevels that bound to BoNT/A and to synthetic BoNT/A peptides (FIG. 18).In their binding to active BoNT/A, BALB/c protective antisera had up to36-fold higher amounts of IgG antibodies relative to non-protectiveantisera (FIG. 18). Similarly, for SJL, the protective antibodies had upto 16-fold higher levels of IgG that bound to active BoNT/A than did thenon-protective antibodies (FIG. 19). Furthermore, non-protective SJL andBALB/c antibodies each exhibited little or no binding to the peptides.These results demonstrate that the major difference between theprotective and non-protective antibodies was the fact thatnon-protective antibodies, obtained after only one immunization withBoNT/A, were primarily of the IgM class. In contrast, protectiveantibodies obtained 10 days after the first booster displayed anIgM-to-IgG switch. In sum, these results indicate that protection isassociated with antibodies of the IgG class.

EXAMPLE IX Mapping of the H Chain Recognition Profile in Antisera from aCohort of Cervical Dystonia (CD) Patients

This example demonstrates that an in vitro assay can be used todetermine amounts of blocking or protective antibodies against BoNT/A inhuman serum samples. This example further demonstrates that acombination assay using, for example, two or three synthetic BoNT/Apeptides can be used for sensitive detection of the presence of specificanti-toxin antibodies in, for example, BOTOX® treated patients.

A. Methods for Data Analysis

MPA-positive cervical dystonia (CD) serum samples were obtained fromAllergan, Parkinson's Disease Center and Movement Disorders Clinic ofBaylor College of Medicine, and the Arizona Dystonia Institute. CDpatient sera protected against a lethal dose of BoNT/A in a mouseprotection bioassay were screened with 60 synthetic toxin peptidescorresponding to the entire H chain of BoNT/A (FIG. 1). The IgG fractionof hyperimmune sera of human volunteers (obtained from the Department ofthe Army) against pentavalent toxin (BoNT/A, B, C, D and E) was used asa positive control. An aliquot (50 μl) of each of the 60 syntheticoverlapping peptides, dissolved in 0.01 M phosphate buffer, pH 7.2containing 0.15 M NaCl (1.0 μg/50 μl of PBS), was added to three wellsof a flexible polyvinyl chloride 96-well plate. Peptides were allowed tobind for two hours at 37° C. followed by overnight incubation at 4° C.Plates were washed five times with PBS to remove unbound peptide andthen blocked for one hour at 37° C. with 0.5% bovine serum albumin inPBS (BSA/PBS). An appropriate volume of each of the mouse protectionassay (MPA)-positive CD sera was preincubated with an equal volume ofTeNT toxoid (1 mg/ml) for three hours at 37° C. after which it wasdiluted to 1:500 (vol/vol) with 0.1% BSA/PBS, pipetted (50 μl) intopeptide-coated wells and allowed to react for three hours at 37° C.followed by further incubation overnight at 4° C. After washing thewells five times with PBS, 50 μl of prediluted (1:500 vol/vol, in 0.1%BSA/PBS) immunoglobulin fraction of rabbit anti-human IgG (DAKOCorporation; Carpinteria, Calif. A0424)+IgM (Mu chain; DAKO, A0426) wasadded and allowed to react at 37° C. for two hours. The wells werewashed five times with PBS followed by addition of 50 μl of ¹²⁵I-ProteinA (2×10⁵ CPM in 0.1% BSA/PBS) to each well and incubation for two hoursat room temperature. Finally, plates were washed thoroughly to eliminateunbound radioactivity; individual wells were cut out and transferredinto separate tubes; and the incorporated radioactivity was counted in agamma-counter (1277 Gamma Master; LKB, Finland). The results, which wereobtained from triplicate analyses, were expressed as the ratios of meanCPM bound by peptides over CPM bound by control peptides or bovine serumalbumin (BSA).

For determining antibody binding to BoNT/A or BoNT/B, triplicate wellswere coated with the appropriate inactive BoNT/A or BoNT/B toxin (0.5μg/50 μl of PBS). A similar procedure was then used to determine theamount of antibody bound by BoNT/A or BoNT/B using human MPA-positive CDsera pre-absorbed with TeNT.

B. Assay of Total Antibodies Bound to BoNT/A and BoNT/B

Due to varying amounts of anti-TeNT antibodies in human sera and thecross-reactivity of these antibodies with both BoNT/A as well as BoNT/B(Behzod et al., Immunol. Invest. 31:247-262 (2002)), the binding assaydescribed above was modified. Essentially, the reaction with BoNT/A orsynthetic BoNT/A peptides was carried out either after absorption of thesera with TeNT or, more conveniently, in the presence of a large excessof TeNT as described further below. The pool of positive controlantisera was obtained from human volunteers, and was tested at twodilutions (1:1000 and 1:2000, vol/vol).

Binding studies of the antisera from CD patients as well as sera fromunimmunized controls showed that the sera had different levels ofnon-specific binding to unrelated protein (BSA) and peptides. This highnon-specific binding affected both the net cpm values as well as theratio of the signal (specific binding) to background (non-specificbinding). Sera from the same cervical dystonia patients prior to toxintreatment (pre-immune sera) were not available to correct for thenon-specific binding. However, the amount of radiolabel bound by certainsynthetic H peptides was observed to be essentially the same as theamount of radiolabel bound to unrelated proteins and peptides. Thesenon-antibody-binding H chain peptides (for example, N2, N3, N5, N6, N7,N9, N10, N11, N12, etc.; see FIG. 1) were utilized as an internalcontrol for each serum. In particular, binding was expressed for eachserum as a ratio of the amount of antibody bound by a test peptide overthe average of the amounts of antibody bound by four of the non-antibodybinding H-chain peptides (N2, N12, C17 and C23). The value for such aratio of antibodies bound to a given peptide from a given serum wasessentially constant.

In assays to determine the total amounts of antibody present in CDpatient sera, BSA and the four non-binding peptides N2, N12, C17 and C23were used as negative controls. The results of antibody binding toBoNT/A toxoid in 28 MPA-positive CD sera and 10 human sera fromunimmunized controls are summarized in FIG. 21. The results show that 27out of 28 (96.4%) MPA-positive sera bound antibody levels that wereclearly higher than those bound by the controls. These results validatethe use of assays performed with human sera in a large excess of TeNT todetermine the total amounts of antibodies to BoNT/A present in the serumof a patient in the course of treatment with BOTOX®.

In determining the total amount of anti-BoNT/B antibodies present in CDpatient sera, BSA was used as the negative control. The results ofbinding to the BoNT/B toxoid of antibodies in 28 MPA-positive CD seraand 10 human sera from unimmunized controls are summarized in FIG. 22.The results show that 27 out of 28 (96.4%) of MPA-positive sera boundantibody levels that were clearly higher than those bound by thecontrols, while one was close to the borderline. These results validatethe use of this assay for determining total amounts of antibodies toBoNT/B present in patient serum in the course of treatment with a BoNT/Bformulation.

C. Mapping of Epitopes Recognized by Antibodies in of MPA-Positive Seraof Cervical Dystonia Patients

The results of mapping by the synthetic H-chain peptides of antibodiesfrom 28 CD patients that were MPA positive are shown in FIGS. 24 to 26and summarized in Table 6. These data, which represent four replicateexperiments, are compared to binding profiles obtained with hyperimmunehuman sera at 1:1000 and 1:2000 (vol/vol). As described above, theresults in FIGS. 24 to 26 and in Table 6 are based on the ratio of cpmbound by a given peptide/cpm bound by BSA and/or average of cpm bound bypeptides N2, N12, C17 and C23. In Table 6, (−) denotes no detectablebinding; (±) indicates very low but reproducible binding (ratio ofspecific over non-specific binding of 1.61-2.0); and different numbersof (+) signs indicate different levels of binding. As can be seen bycomparison with the data reported above, peptides which bound antibodiesin the sera of the CD patients also bound antibodies within hyperimmunesera. However, not every peptide that bound antibodies in hyperimmuneserum was able to bind antibodies in patient sera, indicating that theantibody-binding profile of the patients' sera was more restricted thanthe profile of the hyperimmune sera.

Furthermore, variability was seen among the binding profiles fordifferent patients. As an example, the antisera of some patients boundpeptide N4, whereas other sera had no such binding-activity. Thisinter-patient variability is consistent with the fact that immuneresponses to protein antigens are known to be under genetic control andthat the response to each epitope within a protein is under separategenetic control (Okuda et al., J. Immunol. 121:866-868 (1978)).

Significantly, however, some peptides bound antibodies in most of thepatients. For example, 25 out of 28 sera contained antibodies that boundto peptide N25, although the amounts bound varied from patient topatient with three sera (patients 45, 304 and 310) having marginallevels of antibodies to this peptide. Peptide C10 bound antibodies insera of 24 out of 28 patients, with the sera of patients 43, 45, 53 andSD displaying very low (±) or no (−) antibody binding to peptide C10.Peptide C15 displayed low (+) to medium (++) binding to antibodies in 17patient sera; very low (±) binding with nine patient sera; and nobinding with two sera. The antibody-binding activity of peptide C20 wasgenerally lower than peptides N25 or C10 but was low (+) to high (+++)in nine of the patient sera, while eight sera showed no antibodybinding, and 11 sera showed very low (±), but reproducible, levels ofbinding. In addition, peptide C31 bound antibodies in 17 sera, showedvery low binding in eight patient sera, and displayed no detectableantibody binding with three patient sera. These results indicate that,while there is some peptide-binding variability among MPA-positive CDpatient sera, several synthetic BoNT/A peptides bind antibodies in thelarge majority of patient sera.

D. Synthetic Peptide Assay for Analysis of Reactivity of MPA-PositivePatient Sera

As disclosed above, MPA-positive cervical dystonia patient seracontained antibodies that bound to one or more of the peptides N25, C10,C15, C20 and C31, indicating that binding to one or more of thesepeptides can used to determine the presence of antibody responses inpatient sera. FIGS. 27, 28, 29 and 30 show the ratio of the specific cpmbound in the same assay to non-binding peptides and to BSA. As shown inFIG. 26, analysis on the basis of peptide N25 was able to distinguishclearly 21 out of 28 (75%) of patient sera from unimmunized controls.Binding to peptide C10 was also able to distinguish 21 out of 28 serafrom the controls, while binding to peptides C15 and C31 distinguished18 (64.3%) and 20 (71.4%) out of 28 sera, respectively (FIGS. 28 to 30).

Combinations of two or more peptides were also assayed for theirdiscriminatory capability. As shown in FIG. 30, when peptides N25 andC10 were combined in the assay, 25 out of 28 (89.3%) of the CD sera werediscriminated from controls. The combination of peptides N25, C10 andC31 distinguished 24 out of 28 sera (85.7%; FIG. 31), and thecombination of peptides N25, C10 and C15 distinguished 25 out of 28(89.3%) of the MPA-positive CD sera from controls (see FIG. 32).Finally, a combination of four peptides (N25, C10, C15 and C31)distinguished 21 out of 28 sera (75%) from the controls, as shown inFIG. 33. These results demonstrate that a combination assay usingpeptides N25 and C10 or N25, C10 and C15 can be useful for detecting thepresence of specific anti-toxin antibodies in BOTOX® treated patients.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention.

What is claimed:
 1. A tolerogizing method of preventing or reducingimmunoresistance to botulinum toxin therapy in an individual, the methodcomprising the step of administering to said individual a tolerogizingcomposition; wherein said tolerogizing composition comprising atolerogizing agent conjugated to a BoNT/A peptide, said BoNT/A peptideis at most 60 amino acids in length and comprises amino acids 771-789 ofSEQ ID NO: 1, amino acids 785-803 of SEQ ID NO: 1, a conservative BoNT/Aamino acid sequence variant thereof, or a tolerogenic BoNT/A amino acidsequence fragment thereof; wherein said amino acids 771-789 of SEQ IDNO: 1, amino acids 785-803 of SEQ ID NO: 1, a conservative BoNT/A aminoacid sequence variant thereof and a tolerogenic BoNT/A amino acidsequence fragment thereof produce a decrease in a humoral immunologicalresponse to a botulinum toxin antigen thereby inducing a specificimmunological tolerance to the botulinum toxin antigen; wherein saidconservative BoNT/A amino acid sequence variant producing a decreasedimmunological response comprises 1-4 conservative amino acidsubstitutions to amino acids 771-789 of SEQ ID NO: 1, or 1-4conservative amino acid substitutions to amino acids 785-803 of SEQ IDNO: 1; wherein said tolerogenic BoNT/A amino acid sequence fragmentproducing a decreased immunological response comprises at least sixconsecutive amino acids of 771-789 of SEQ ID NO: 1, or at least sixconsecutive amino acids of 785-803 of SEQ ID NO: 1; and whereinadministration of said tolerogizing composition decreases animmunological response to a botulinum toxin antigen.
 2. The tolerogizingmethod of claim 1, wherein said tolerogizing composition is administeredto said individual prior to receiving botulinum toxin therapy, during acourse of botulinum toxin therapy, or after onset of immunoresistance tobotulinum toxin therapy.
 3. The tolerogizing method of claim 1, whereinsaid individual is at increased risk for immunoresistance to a botulinumtoxin therapy.
 4. The tolerogizing method of claim 1, wherein saidbotulinum toxin antigen is a botulinum toxin type A antigen.
 5. Thetolerogizing method of claim 1, wherein said decreased immunologicalresponse is a decrease in a T-cell response.
 6. The tolerogizing methodof claim 5, wherein said decrease T-cell response is a decrease inT-cell proliferation or a decrease in T-cell cytokine secretion.
 7. Thetolerogizing method of claim 1, wherein said decreased immunologicalresponse is a decrease in an anti-botulinum toxin antibody titer.
 8. Thetolerogizing method of claim 7, wherein said anti-botulinum toxinantibody titer is an anti-botulinum toxin type A antibody titer.
 9. Thetolerogizing method of claim 1, wherein said tolerogizing composition isprepared as a pharmaceutical composition.
 10. The tolerogizing method ofclaim 1, wherein said BoNT/A peptide comprises amino acids 771-789 ofSEQ ID NO: 1, or amino acids 785-803 of SEQ ID NO:
 1. 11. Thetolerogizing method of claim 1, wherein said BoNT/A peptide comprisesamino acids 785-803 of SEQ ID NO: 1, a conservative BoNT/A amino acidsequence variant thereof, or a tolerogenic BoNT/A amino acid sequencefragment thereof; wherein said conservative BoNT/A amino acid sequencevariant producing a decreased immunological response comprises 1-4conservative amino acid substitutions to amino acids 785-803 of SEQ IDNO: 1; and wherein said tolerogenic BoNT/A amino acid sequence fragmentproducing a decreased immunological response comprises at least sixconsecutive amino acids of 785-803 of SEQ ID NO:
 1. 12. The tolerogizingmethod of claim 11, wherein said BoNT/A peptide comprises amino acids785-803 of SEQ ID NO:
 1. 13. The tolerogizing method of claim 1, whereinsaid BoNT/A peptide comprises amino acids 771-789 of SEQ ID NO: 1, aconservative BoNT/A amino acid sequence variant thereof, or atolerogenic BoNT/A amino acid sequence fragment thereof; wherein saidconservative BoNT/A amino acid sequence variant producing a decreasedimmunological response comprises 1-4 conservative amino acidsubstitutions to amino acids 771-789 of SEQ ID NO: 1; and wherein saidtolerogenic BoNT/A amino acid sequence fragment producing a decreasedimmunological response comprises at least six consecutive amino acids of771-789 of SEQ ID NO:
 1. 14. The tolerogizing method of claim 13,wherein said BoNT/A peptide comprises amino acids 771-789 of SEQ IDNO:
 1. 15. The tolerogizing method of claim 1, wherein said BoNT/Apeptide is at most 40 amino acids in length.
 16. The tolerogizing methodof claim 1, wherein said BoNT/A peptide is at most 25 amino acids inlength.
 17. The tolerogizing method of claim 1, wherein saidtolerogizing agent is a polyethylene glycol.
 18. The tolerogizing methodof claim 1, wherein said tolerogizing agent is a monomethoxypolyethyleneglycol.
 19. The tolerogizing method of claim 1, wherein saidtolerogizing agent is a polyvinyl alcohol.